Recombinant Newcastle disease viruses useful as vaccines or vaccine vectors

ABSTRACT

The present invention concerns an antigenomic RNA of Newcastle Disease virus (NDV) carrying one or more foreign genes inserted before NP gene, between P and M genes, and/or between HN and L genes. The invention is also directed toward a cDNA encoding a recombinant antigenomic RNA having one or more foreign genes inserted according to the invention, a cell containing the cDNA, a plasmid comprising the cDNA, a cell containing the plasmid, a cell containing the recombinant antigenomic RNA, and a recombinant NDV containing the recombinant antigenomic RNA of the invention, such as a recombinant NDV carrying one or more foreign genes recovered from transcription of the cDNA or the plasmid in a competent cell. The recombinant NDV carrying the one or more foreign genes can be used as a vaccine or vaccine vector.

[0001] The present patent application is a continuation-in-partapplication of pending U.S. patent application Ser. No. 09/926,431 filedon Mar. 6, 2002, which is a U.S. national phase entry application under35 U.S.C. §371 based on PCT/US00/06700 filed on May 5, 2000. The presentpatent application also claims the benefit of U.S. Provisional PatentApplication No. 60/381,462 filed on May 17, 2002. The disclosures ofapplication Ser. No. 09/926,431 and 60/381,462 are hereby incorporatedby reference.

[0002] The present application relates to recombinant Newcastle diseaseviruses carrying one or more foreign genes, i.e. genes not foundnaturally in the Newcastle disease virus, which are useful as vaccinesor vaccine vectors.

BACKGROUND OF THE INVENTION

[0003] Newcastle disease is a highly contagious viral disease affectingall species of birds. The disease can vary from an asymptomaticinfection to a highly fatal disease, depending on the virus strain andthe host species. Newcastle disease has a worldwide distribution and isa major threat to the poultry industries of all countries. Based on theseverity of the disease produced in chickens, Newcastle disease virus(NDV) strains are grouped into three main pathotypes: lentogenic(strains that do not usually cause disease in adult chickens), mesogenic(strains of intermediate virulence) and velogenic (strains that causehigh mortality).

[0004] NDV is a member of the genus Rubulavirus in the familyParamyxoviridae. The genome of NDV is a non-segmented, single-stranded,negative-sense RNA of 15186 nucleotides (Krishnamurthy & Samal, 1998;Phillips et el., 1998; de Leeuw & Peeters, 1999). The genomic RNAcontains six genes that encode the following proteins in the order of:the nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M),fusion protein (F), haemagglutinin-neuramimidase (HN) and largepolymerase protein (L). Two additional proteins, V and W, of unknownfunction are produced by RNA editing during P gene transcription(Steward et al., 1993). A schematic diagram of the genetic map of NDVgenomic RNA is shown in FIG. 1.

[0005] Three proteins, i.e. NP, P and L proteins, constitute thenucleocapsid. The genomic RNA is tightly bound by the NP protein andtogether with the P and L proteins form the functional nucleocapsidwithin which resides the viral transcriptive and replicative activities.The F and HN proteins form the external envelope spikes, where the HNglycoprotein is responsible for attachment of the virus to host cellreceptors and the F glycoprotein mediates fusion of the viral envelopewith the host cell plasma membrane thereby enabling penetration of theviral genome into the cytoplasm of the host cell. The HN and F proteinsare the main targets for the immune response. The M protein forms theinner layer of the virion.

[0006] NDV follows the general scheme of transcription and replicationof other non-segmented negative-strand RNA viruses. The polymeraseenters the genome at a promoter in the 3′ extragenic leader region andproceeds along the entire length by a sequential stop-start mechanismduring which the polymerase remains template bound and is guided byshort consensus gene start (GS) and gene end (GE) signals. Thisgenerates a free leader RNA and six non-overlapping subgenomic mRNAs.The abundance of the various mRNAs decreases with increasing genedistance from the promoter. The genes are separated by short intergenicregions (1-47 nucleotides) which are not copied into the individualmRNAs. RNA replication occurs when the polymerase somehow switches to aread-through mode in which the transcription signals are ignored. Thisproduces a complete encapsulated positive-sense replicative intermediatewhich serves as the template for progeny genomes.

[0007] Reverse-genetic techniques have been reported to recovernegative-sense viruses from cloned cDNA (Conzelmann, 1996). For NDV,reverse-genetic technology is currently available for avirulent strainLaSota (Romer-Oberdorfer et al., 1999; Peeters et al., 1999).

SUMMARY OF THE INVENTION

[0008] Reverse-genetic techniques were used in making the recombinantNDV of the present invention from cloned cDNA. This approach involvesco-expression of the cloned cDNA of full length NDV genome andnucleocapsid proteins (the NP, P and L proteins) from transfectedplasmids using the vaccinia virus/T7 RNA polymerase expression system.

[0009] Within the scope of the present invention, recombinant NDV can berecovered from cDNA and the genome of NDV can be manipulated at the cDNAlevel. The production of infectious NDV from cloned cDNA can be used toengineer NDV carrying foreign genes. With the manipulation of the genomeof NDV, one can insert foreign sequences into the NDV genome forco-expression. For example, the gene for protective antigen of anotheravian pathogen or the genes for avian cytokines can be inserted into theNDV genome for co-expression. Thus, the present invention includesmultivalent genetically engineered NDV vaccines carrying genes encodingimmunogens (e.g. immunogenic proteins) for influenza virus, infectiousbursal disease virus, rotavirus, infectious bronchitis virus, infectiouslaryngotracheitis virus, chicken anemia virus, Marek's disease virus,avian Leukosis virus, avian adenovirus and avian pneumovirus.

[0010] The present invention also is directed toward a geneticallyengineered NDV carrying avian cytokine genes. A NDV carrying at leastone gene encoding an avian cytokine, e.g. an interleukin such as IL-2and IL-4, can be used as a vaccine.

[0011] The recombinant NDV prepared by insertion of foreign genes intothe NDV genome can express the foreign genes in cells infected by therecombinant NDV. As a result, the recombinant NDV can be used to expressproteins of non-avian pathogens or other avian pathogens. Therefore, therecombinant NDV can be used as a vaccine vector.

[0012] One of the objects of the present invention is a recombinantantigenomic RNA of Newcastle disease virus, comprising NP gene, P gene,M gene, F gene, HN gene and L gene in this order from a 5′ to 3′direction, said antigenomic RNA further comprising n foreign nucleotidecomplexes inserted (a) before the NP gene, (b) between the P and Mgenes, and/or (c) between the HN and L genes, wherein n is 1, 2, 3 or 4;

[0013] each of the foreign nucleotide complexes comprising a Newcastledisease virus gene start sequence, an open reading frame of a foreigngene and a Newcastle disease virus gene end sequence in this order fromthe 5′ to 3′ direction, wherein the foreign gene is a gene not foundnaturally in the Newcastle disease virus;

[0014] wherein when n is 2, 3 or 4, the foreign nucleotide complexes arethe same or different; and

[0015] wherein when 2, 3 or 4 foreign nucleotide complexes are insertedtogether before the NP gene, between the P and M genes, or between theHN and L genes, the foreign nucleotide complexes are sequentially linkeddirectly or indirectly.

[0016] Since each foreign nucleotide complex has a NDV gene startsignal, i.e. GS sequence motif, upstream of the open reading frame (ORF)of the foreign gene and a NDV gene end signal, i.e. GE sequence motif,downstream of the ORF of the foreign gene, each foreign nucleotidecomplex forms a transcriptional unit.

[0017] The recombinant antigenomic RNA of NDV of the present inventionpreferably further comprises NP-P intergenic region between the NP geneand P gene, P-M intergenic region between the P gene and M gene, M-Fintergenic region between the M gene and F gene, F-HN intergenic regionbetween the F gene and HN gene, and/or HN-L intergenic region betweenthe HN gene and L gene. More preferably, the recombinant antigenomic RNAof NDV of the present invention further comprises NP-P intergenic regionbetween the NP gene and P gene, P-M intergenic region between the P geneand M gene, M-F intergenic region between the M gene and F gene, F-HNintergenic region between the F gene and HN gene, and HN-L intergenicregion between the HN gene and L gene. When one or more of the foreignnucleotide complexes are inserted between the P and M genes, the foreignnucleotide complexes can be inserted into the P-M intergenic region ifpresent. Similarly, when one or more of the foreign nucleotide complexesare inserted between the HN and L genes, the foreign nucleotidecomplexes can be inserted into the HN-L intergenic region. Optionally,one or more of the NP-P intergenic region, P-M intergenic region, M-Fintergenic region, F-HN intergenic region, and HN-L intergenic regionare replaced with a single nucleotide, dinucleotide or anoligonucleotide of 3-80 nucleotides (preferably 4-60 nucleotides) inlength, wherein the oligonucleotide optionally contains one or morerestriction sites.

[0018] When one or more of the foreign nucleotide complexes are insertedbefore the NP gene, the foreign nucleotide complexes preferably areinserted into a non-coding region immediately before the ORF of the NPgene, so that the ORF of the foreign gene in each of the foreignnucleotide complexes is flanked by NDV gene start and gene end signalsand the ORF of the NP gene is preceded by a NDV gene start signal, withthe GS-foreign gene ORF-GE structure preceding the GS signal for the NPORF.

[0019] Within the scope of the invention is a recombinant antigenomicRNA of NDV having one or more foreign nucleotide complexes insertedbetween P and M genes. The antigenomic RNA can be made by inserting theone or more foreign nucleotide complexes into the noncoding region of Pgene after the stop codon, but before the NDV gene end signal of the Pgene. When only one foreign nucleotide complex is inserted into thenoncoding region of P gene after the stop codon, the ORF of the foreigngene is preceded by a NDV gene end and NDV gene start signals, resultingin the ORF of the P gene being preceded by a NDV gene end signal, whichis followed by a NDV gene start signal, the ORF of the foreign gene, anda NDV gene end signal in that order (the ORF of the following M gene ispreceded by a NDV gene start signal). More foreign gene complexes can beinserted after this foreign gene complex. Similarly, the recombinantantigenomic RNA of NDV having one or more foreign nucleotide complexesinserted between P and M genes can be made by inserting the one or moreforeign nucleotide complexes into the noncoding region of M gene beforethe ORF of the M gene.

[0020] The present invention is also directed toward a process ofpreparing the recombinant antigenomic RNA of the invention, comprisingthe following steps:

[0021] (i) providing a cDNA comprising NP gene, P gene, M gene, F gene,HN gene and L gene in this order, said cDNA further comprising n foreignnucleotide complexes inserted (a) before the NP gene, (b) between the Pand M genes, and/or (c) between the HN and L genes, wherein n is 1, 2, 3or 4;

[0022] each of the foreign nucleotide complexes comprising a Newcastledisease virus gene start sequence, an open reading frame of a foreigngene and a Newcastle disease virus gene end sequence in this order fromthe 5′ to 3′ direction, wherein the foreign gene is a gene not foundnaturally in the Newcastle disease virus;

[0023] wherein when n is 2, 3 or 4, the foreign nucleotide complexes arethe same or different; and

[0024] wherein when 2, 3 or 4 foreign nucleotide complexes are insertedtogether before the NP gene, between the P and M genes, or between theHN and L genes, the foreign nucleotide complexes are sequentially linkeddirectly or indirectly;

[0025] (ii) transcribing the antigenomic cDNA to form a mixturecontaining an antigenomic RNA; and thereafter

[0026] (iii) isolating the antigenomic RNA.

[0027] In some embodiments of the process of preparing the recombinantantigenomic RNA of the invention, the cDNA used in step (i), comprisingNP gene, P gene, M gene, F gene, HN gene and L gene having the n foreignnucleotide complexes inserted, is prepared by (I) constructing a cDNAcomprising the NP gene, P gene, M gene, F gene, HN gene and L gene inthis order; and thereafter (II) inserting the n foreign nucleotidecomplexes (a) before the NP gene, (b) between the P and M genes, and/or(c) between the HN and L genes. Preferably, the cDNA constructed in step(I) and/or the cDNA constructed in step (II) are in a plasmid, such aspBR322 or pGEM-7Z. In step (ii), the cDNA preferably is transcribed incells expressing a RNA polymerase, such as T7 RNA polymerase.

[0028] The present invention is also directed toward a recombinant NDVcomprising a recombinant antigenomic RNA carrying one or more foreigngenes of the present invention. The recombinant NDV can be produced by aprocess comprising the following steps:

[0029] (i) providing cells capable of synthesizing T7 RNA polymerase;

[0030] (ii) transfecting the cells with a plasmid comprising the cDNAencoding the antigenomic RNA having one or more foreign genes insertedaccording to the invention, a plasmid encoding NP protein, a plasmidencoding P protein, and a plasmid encoding L protein to obtaintransfected cells in a medium; and thereafter

[0031] (iii) isolating Newcastle disease virus from a supernatant of themedium of step (ii) to obtain the recombinant Newcastle disease virus.

[0032] The cells capable of synthesizing T7 RNA polymerase provided instep (i) can be animal cells of an avian or mammalian species, plantcells, or cells from a cell line expressing T7 RNA polymerase.

[0033] Within the scope of the present invention are a cDNA encoding arecombinant antigenomic RNA having one or more foreign genes insertedaccording to the invention, a cell containing the cDNA, a plasmidcomprising the cDNA, a cell containing the plasmid, a cell containingthe recombinant antigenomic RNA, and a recombinant NDV containing therecombinant antigenomic RNA of the invention, e.g. a recombinant NDVcarrying one or more foreign genes recovered from transcription of thecDNA or the plasmid in a competent cell. The recombinant NDV containingthe recombinant antigenomic RNA of the invention is preferablysubstantially purified. Also preferred is a substantially purifiedrecombinant antigenomic RNA of NDV carrying one or more foreign genesprepared according to the invention.

[0034] The present invention also includes a method of vaccinating anavian animal against Newcastle disease, wherein the avian animal is inneed of the vaccination, comprising administering an effective amount ofthe recombinant NDV carrying one or more foreign genes according to theinvention to the avian animal.

[0035] One of the objects of the inventions is a method of treating anavian animal with an avian cytokine, wherein the avian animal is in needof the treatment, said method comprising administering an effectiveamount of the recombinant NDV of the invention carrying one or moreforeign genes encoding one or more avian cytokines, such as avianinterleukins (preferably IL-2 and/or IL-4) to the avian animal.

[0036] Another aspect of the invention is a method of immunizing anavian animal against an avian pathogen selected from the groupconsisting of influenza virus, infectious bursal disease virus,rotavirus, infectious bronchitis virus, infectious laryngotracheitisvirus, chicken anemia virus, Marek's disease virus, avian Leukosisvirus, avian adenovirus and avian pneumovirus, wherein the avian animalis in need of the immunization, said method comprising administering aneffective amount of the recombinant NDV of the invention to the aviananimal, wherein the recombinant NDV carries one or more foreign genesencoding one or more immunogenic proteins of the avian pathogen againstwhich the avian animal is immunized.

[0037] Also within the scope of the invention is a method of immunizinga mammal against a non-avian pathogen, wherein the mammal is in need ofthe immunization, said method comprising administering an effectiveamount of the recombinant NDV of the invention to the mammal, whereinthe recombinant NDV carries one or more foreign genes encoding one ormore immunogenic proteins of the non-avian pathogen, e.g. influenzavirus, SARS-causing virus, human respiratory syncytial virus, humanimmunodeficiency virus, hepatitis A virus, hepatitis B virus, hepatitisC virus, poliovirus, rabies virus, Hendra virus, Nipah virus, humanparainfluenza 3 virus, measles virus, mumps virus, Ebola virus, Marburgvirus, West Nile virus, Japanese encephalitis virus, Dengue virus,Hantavirus, Rift Valley fever virus, Lassa fever virus, herpes simplexvirus and yellow fever virus, against which the mammal is immunized.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038]FIG. 1 is a schematic of the genetic map of NDV genomic RNA.

[0039]FIG. 2 is a map of the genome of NDV strain Beaudette C, whereinthe nucleotide sequence of the NP region is available from the GenBankdatabase with the accession number AF064091. In FIG. 2, the lastnucleotide of the gene end, the first nucleotide of the gene start andthe first and last nucleotides of the leader and trailer are numbered.The gene start and gene end sequences were derived from publishedsources of the NDV.

[0040]FIG. 3 shows the assembly of full-length cDNA of NDV strainLaSota. Seven subgenomic cDNA fragments generated by high-fidelityRT-PCR were assembled in pBR322/dr (not to scale). The numbers above thecDNA in FIG. 3 are the first nucleotide positions of various restrictionsites. Plasmid pBR322 was modified to include a 72 nt oligonucleotidelinker between the EcoRI and PstI sites, an 84 nt hepatitis delta virus(HDV) antigenome ribozyme sequence and a T7 RNA polymerasetranscription-termination signal. Transcription of the plasmid pLaSotaby T7 RNA polymerase resulted in NDV antigenomic RNA with threenon-viral G residues at the 5′ terminus.

[0041]FIG. 4 shows the construction of pLaSota/CAT. An 18 nt fragmentcontaining a PmeI site was inserted into the non-coding regionimmediately before the NP ORF. The ORF of the CAT gene was amplified byPCR with PmeI-tagged primers, digested with PmeI and introduced into thenewly created PmeI site of the NP gene. A set of NDV gene-end (GE) andgene-start (GS) signals normally connected to the NP-P intergenicsequence was placed at the end of the CAT gene. The resulting plasmidpLaSota/CAT gave rise to an antigenomic RNA of 15900 nt, which is amultiple of six.

[0042]FIG. 5 shows plaques produced by rLaSota and rLaSota/CAT on DF1cells. Infected cells overlaid with 1% methylcellulose were incubatedfor a period of 4 days. Plaques were visualized by immunostaining usinga monoclonal antibody against the NDV HN protein.

[0043]FIG. 6 shows the identification of genetic markers in the genomeof rLaSota and confirmation of the presence of the CAT gene in thegenome of rLaSota/CAT. RT-PCR was performed from genomic RNA extractedfrom purified viruses. (a) Identification of genetic markers in thegenome of rLaSota. Primers spanning the corresponding regions were usedfor PCR and the products were subjected to restriction enzyme digestion.Wild-type LaSota was used as control. (b) Confirmation of the presenceof the CAT gene in the recovered rLaSota/CAT by PCR with specificprimers. The larger RT-PCR product (23 kb) from rLaSota/CAT confirmedthe presence of the CAT gene compared with the smaller RT-PCR product(16 kb) from rLaSota.

[0044]FIG. 7 shows a comparison of CAT expression by a recombinant virus(rBC/CAT) recovered from a cDNA encoding a recombinant antigenomic RNAof NDV Beaudefte C having a CAT gene inserted between HN and L genes asprepared in Example 2 and a recombinant virus (rLaSota/CAT) recoveredfrom a cDNA encoding a recombinant antigenomic RNA of NDV LaSota havinga CAT gene inserted before NP gene as prepared in Example 1 at passage6. The CAT gene was expressed from the sixth position in rBC/CAT whileit was expressed from the first position in rLaSota/CAT. Equal cellequivalents of lysates were analysed for acetylation of[¹⁴C]chloramphenicol as visualized by thin-layer chromatography.Relative CAT activity was quantified by densitometry.

[0045]FIG. 8 shows results of Northern blot hybridization blothybridization of intracellular mRNAs encoded by rLaSota and rLaSota/CAT.Poly(A)+ mRNAs were isolated from total intracellular RNA by oligo(dT)chromatography and were electrophoresed on formaldehyde-agarose gels.The gels were transferred to nitrocellulose membrane and probed with thenegative-sense riboprobes.

[0046]FIG. 9 shows multi-step growth curves for wt LaSota (), rLaSota(▪) and rLaSota/CAT (▴) in DF1 cells. Cell monolayers in 25 cm2 flaskswere infected with 0·005 p.f.u. per cell with three replicate flasks pervirus. Samples were taken every 8 h for 56 h. The virus in thesupernatant was titrated by plaque assay. The log titer was derived fromthe mean virus titer and error bars indicate standard deviations.

[0047]FIG. 10 shows the construction of a full-length cDNA to the genomeof NDV strain Beaudette C in a plasmid.

[0048]FIG. 11 shows a full-length NDV cDNA assembled in pBR322/dr fromsubgenomic cDNA fragments (I to VIII) that were generated byhigh-fidelity RT-PCR (not to scale). The blocked arrows indicate theprimers used in RT-PCR. The numbers shown above the primers representthe position in the genome of the first nucleotide of the primer,represented 5′ to 3′. Plasmid pBR322/dr is the modified form of plasmidpBR322, designed to include a 72-nucleotide oligonucleotide linkerbetween the EcoRI and PstI sites, an 84-nucleotide hepatitis delta virus(HDV) antigenome ribozyme sequence and a T7 RNA polymerase transcriptiontermination signal. Transcription by the T7 RNA polymerase of theplasmid pNDVf1 resulted in the NDV antigenomic RNA with 3 nonviral Gresidues at the 5′ terminus.

[0049]FIG. 12 shows the construction of a recombinant NDV cDNA thatcontains a foreign gene inserted into the intergenic region between theHN and L genes, wherein the foreign gene means a gene foreign to NDV andin this case the foreign gene is a gene encoding chloramphenicalacetyltransferase (CAT).

[0050]FIG. 13 shows the construction of pNDVf1/CAT by insertion of theCAT gene cDNA into HN and L intergenic region of the plasmid pNDVf1. Thenucleotide sequences represent the oligonucleotide primer used foramplifying the CAT ORF. The resulting PCR product with the AgeI overhangon either end contained the gene start (GS) signal, noncoding (NC)sequence, and gene end (GE) signal. The nucleotide length of theconstruct was maintained as a multiple of six. The RNA encoded bypNDVf1/CAT included the three 5′-terminal nonviral G residuescontributed by the T7 promoter.

[0051]FIG. 14 shows a recombinant vaccinia virus-based transfectionsystem used to recover infectious NDV from a plasmid containing arecombinant cDNA to the genome of NDV.

[0052]FIG. 15 shows the detection of CAT expression after one passage ofinfectious recombinant NDV carrying the CAT gene recovered from therecombinant vaccinia virus-based transfection system of FIG. 14, whereinthe recombinant NDV resulted from transcription of the recombinant NDVcDNA constructed in FIG. 12.

[0053]FIG. 16 depicts schematically the construction of a recombinantcDNA encoding an antigenome of NDV strain Beaudette C having a foreigngene, a gene encoding GFP, inserted into the noncoding region of P geneby creating a XbaI site via mutation of a TCTCGC segment (nucleotidepositions 3182-3187) forming a TCTAGA segment in the P gene noncodingregion after a stop codon. TTAGAAAAAA represents a cDNA fragment for aNDV gene end signal and ACGGGTAGMAA represents a cDNA fragment for a NDVgene start signal.

DETAILED DESCRIPTION OF THE INVENTION

[0054] In some embodiments of the recombinant antigenomic RNA of thepresent invention, n is 2, 3 or 4 (preferably 2 or 3, and morepreferably 2) and the foreign nucleotide complexes are different. Insome embodiments of the recombinant antigenomic RNA, n is 2, 3 or 4(preferably 2 or 3, and more preferably 2) and the foreign nucleotidecomplexes are the same. In still some embodiments of the recombinantantigenomic RNA, n is 1 or 2.

[0055] In some of the recombinant antigenomic RNAs of the invention, theORF of each of the foreign genes in inserted the foreign nucleotidecomplexes is no more than about 3000 nucleotides, no more than about2000 nucleotides, no more than about 1500 nucleotides, no more thanabout 1000 nucleotides, no more than about 800 nucleotides, no more thanabout 500 nucleotides, or no more than about 300 nucleotides in length.

[0056] In some of the embodiments of the recombinant antigenomic RNA ofthe present invention, where 2, 3 or 4 foreign nucleotide complexes areinserted together before the NP gene, between the P and M genes, orbetween the HN and L genes, the foreign nucleotide complexes aresequentially linked directly or indirectly, and the foreign nucleotidecomplexes have a combined length of no more than about 5000 nucleotides,no more than about 4000 nucleotides, no more than about 3000nucleotides, no more than about 2000 nucleotides, no more than about1000 nucleotides, or no more than about 800.

[0057] The foreign gene inserted in the recombinant antigenomic RNA ofthe invention preferably encode a substance selected from the groupconsisting of chloramphenical acetyltransferase, GFP, an avian cytokine,and an immunogenic protein of influenza virus, infectious bursal diseasevirus, rotavirus, infectious bronchitis virus, infectiouslaryngotracheitis virus, chicken anemia virus, Marek's disease virus,avian leukosis virus, avian adenovirus, or avian pneumovirus. Theforeign gene may encode an immunogenic protein of a non-avian pathogen,e.g. influenza virus, SARS-causing virus, human respiratory syncytialvirus, human immunodeficiency virus, hepatitis A virus, hepatitis Bvirus, hepatitis C virus, poliovirus, rabies virus, Hendra virus, Nipahvirus, human parainfluenza 3 virus, measles virus, mumps virus, Ebolavirus, Marburg virus, West Nile disease virus, Japanese encephalitisvirus, Dengue virus, Hantavirus, Rift Valley fever virus, Lassa fevervirus, herpes simplex virus and yellow fever virus.

[0058] When more than one foreign gene encoding the avian cytokine isinserted, the foreign genes may encode the same or different aviancytokines, such as avian interleukins, e.g. IL-2 and IL-4.

[0059] Examples of the foreign gene encoding an immunogenic protein ofan avian pathogen are HA or NA gene of influenza virus, VP2 orpolyprotein gene of infectious bursal disease virus, S or S1 gene ofinfectious bronchitis virus, glycoprotein gene of infectiouslaryngotracheitis virus, the complete genome of chicken anemia virus,glycoprotein gene of Marek's disease virus, envelope gene of avianleukosis virus, avian adenovirus, and G or F gene of avian pneumovirus.

[0060] Examples of the foreign gene encoding an immunogenic protein of anon-avian pathogen are HA or NA gene of influenza virus, S or S1 gene ofSARS-causing virus, G or F gene of human respiratory syncytial virus,gp60, gp120 or gp4l gene of human immunodeficiency virus, surfaceantigen gene of hepatitis A virus, surface antigen gene of hepatitis Bvirus, surface antigen of hepatitis C virus, capsid proteins gene ofpoliovirus, G protein gene of rabies virus, G or F protein gene ofHendra virus, G or F protein gene of Nipah virus, HN or F protein geneof human parainfluenza 3 virus, H or F protein gene of measles virus, HNor F protein gene of mumps virus, G protein gene of Ebola virus, Gprotein gene of Marburg virus, envelope protein gene of West Niledisease virus, envelope protein gene of Japanese encephalitis virus,envelope protein gene of Dengue virus, glycoprotein gene of Hantavirus,glycoprotein gene of Rift Valley fever virus, G1 or G2 protein gene ofLassa fever virus, glycoprotein genes of herpes simplex virus, andglycoprotein gene of yellow fever virus.

[0061] The present invention is also directed toward an antigenomic RNAof NDV carrying one or more foreign genes inserted before the NP gene,between the P and M genes, and/or between the HN and L genes, wherein atleast one of the foreign genes encodes a tumor antigen, such as pg100,MAGE1, MAGE3 and CDK4.

[0062] In the recombinant antigenomic RNA of the invention, the foreignnucleotide complexes preferably are inserted before the NP gene, and/orbetween the P and M genes. More preferably, at least one of the foreignnucleotide complexes is inserted before the NP gene. In some embodimentsof the recombinant antigenomic RNA, at least one of the foreignnucleotide complexes is inserted before the NP gene and at least one ofthe foreign nucleotide complexes is inserted between the P and M genes.In some embodiments, at least one of the foreign nucleotide complexes isinserted before the NP gene and at least one of the foreign nucleotidecomplexes is inserted between the HN and L genes. In still someembodiments, at least one of the foreign nucleotide complexes isinserted before the NP gene, at least one of the foreign nucleotidecomplexes is inserted between the P and M genes, and at least one of theforeign nucleotide complexes is inserted between the HN and L genes. Inyet some embodiments, at least one of the foreign nucleotide complexesis inserted between the P and M genes. Most preferably, the foreignnucleotide complexes are inserted only before the NP gene.

[0063] NDV grows to very high titers (<109 PFU/ml) in many cell linesand eggs and elicits strong humoral and cellular immune responses invivo. NDV naturally infects via respiratory and alimentary tract mucosalsurfaces. NDV replicates in the cytoplasm of infected cells and does notundergo genetic recombination, making vaccine vectors based on therecombinant NDV carrying foreign genes stable and safe. Due to thesecharacteristics of NDV described herein, recombinant NDVs that canexpress foreign genes carried in the recombinant NDVs are good vaccines,wherein the foreign genes encode immunogenic proteins of pathogens.

[0064] The recombinant NDV on the invention carrying one or moreinserted foreign genes show robust expression of the foreign genes.Moreover, the recombinant NDV expressing one or more of the foreign genecan replicate in cell culture and in vivo. Avirulent NDV recombinantsexpressing heterologous proteins could be used as multivalent vaccines.

[0065] The recombinant NDV generated from the recombinant antigenomicRNA carrying one or more foreign genes inserted according to theinvention can also be used as an inactivated vaccine.

[0066] The vaccine or vaccine vector based on the recombinant NDVgenerated from the recombinant antigenomic RNA carrying one or moreforeign genes inserted according to the invention can be administeredtopically, via the respiratory route, orally or via an injection. Thedose of the vaccine or vaccine vector to be used can be readilydetermined by a person skilled in the art based on the disease, the hostsubject species, and the age, sex and/or health condition of the hostsubject involved.

EXAMPLE 1

[0067] In this working example, an embodiment of the invention in whichthe recombinant NDV containing CAT as the foreign gene inserted beforethe NP gene was prepared.

[0068] A. Assembly of a Full-Length Clone of NDV Strain LaSota andRecovery of NDV LaSota from a Plasmid

[0069] NDV lentogenic strain LaSota was grown in 10-day-old embryonated,specific-pathogen-free (SPF) eggs. The virus was purified from allantoicfluid as described previously (Kingsbury, 1966). Viral RNA was extractedfrom the purified virus by using TRIzol according to the manufacturer'sprotocol (Life Technologies). The extracted RNA was subjected to RT-PCRwith virus-specific primer pairs (Table 1) to generate seven overlappingPCR fragments of the entire viral genome with high-fidelity Pfx DNApolymerase (Life Technologies). In Table 1, the cDNA fragmentscorrespond to the fragments shown in FIG. 3, wherein the T7 promotersequence is in italics, virus sequences are underlined, restrictionsites are in bold, and the partial HDV ribozyme sequence (24 nt)overhang is shown in lower case. A low-copy-number plasmid, pBR322, wasmodified as pBR322/dr to contain a 72 nt linker between the EcoRI andPstI sites for subsequent assembly of full-length NDV strain LaSota. TheLaSota strain cDNA was placed in the antigenomic orientation between theT7 promoter and a self-cleaving hepatitis delta virus (HDV) antigenomeribozyme, followed by a T7 RNA polymerase terminator (FIG. 3). Twogenetic markers were introduced into the full-length cDNA for thepurpose of identifying the recovered virus. An MluI site was created inthe F-HN intergenic region and a SnaBI site was created in the HN-Lintergenic region. For the construction of NP, P and L expressionplasmids, the open reading frame (ORF) of each gene was amplified fromthe above full-length clone by PCR. The NP gene was cloned in theplasmid PGEM-7Z (Promega) between EcoRI and BamHI sites. The P and Lgenes were cloned in an expression plasmid thathas anencephalomyocarditis virus internal ribosome entry site (IRES)downstream of the T7 RNA polymerase promoter, and they make use of thetranslation start codon contained in the NcoI site of the IRES. Theassembled full-length cDNA clone and the support plasmids encodingLaSota NP, P and L proteins were sequenced in their entirety. Theresulting full-length clone and support plasmids were designatedpLaSota, pNP, pP and pL, respectively.

[0070] A cDNA clone encoding NDV strain LaSota antigenomic RNA wasassembled from seven cDNA fragments, as shown in FIG. 3. This plasmid,termed pLaSota, positioned the NDV cDNA between the T7 promoter and theHDV ribozyme sequence. During its construction, the antigenomic cDNA wasmodified to generate two new restriction sites as markers. A geneticmarker was introduced into the intergenic region between the F and HNgenes by changing two nucleotides to mutate the original AgeI site to aunique MluI site (positions 6292-6297 in the full-length cDNA clone).Similarly, a unique SnaBI site was generated in the HN and L intergenicregion by changing four nucleotides (positions 8352-8357). To facilitatetranscription by T7 RNA polymerase, three G residues were includedbefore the NDV leader sequence.

[0071] In order to recover NDV from the cloned cDNA to the antigenome ofNDV LaSota, transfection was carried out as described here (based on ageneral procedure schematically shown in FIG. 14). HEp-2 cells (6-wellplates) were infected at 1 p.f.u. per cell with modified vaccinia virus(MVA/T7) expressing T7 RNA polymerase. A mixture of a plasmid containingNP gene ORF, a plasmid containing P gene ORF, and a plasmid containing Lgene ORF all under the control of the T7 promoter (25, 15 and 0.5 pg perwell, respectively) and a fourth plasmid encoding the NDV (5 pg) wastransfected with LipofectAMINE Plus (Life Technologies). Four hoursafter transfection, cells were washed and the medium was replaced with 2ml fresh medium (DMEM with 0% foetal calf serum and 1 pg/mlacetyltrypsin). Three days post-transfection, the supernatant washarvested, clarified and used to infect fresh HEp-2 cells. Three dayslater, 100 pl supernatant was taken to inoculate into the allantoiccavity of 10-day-old embryonated SPF eggs. After 96 h, allantoic fluidwas harvested and tested for haemagglutinating (HA) activity. After twopassages in eggs, the virus was plaque-purified to eliminate vacciniavirus. The plaques produced by the virus were stained with monoclonalantibodies specific to the NDV HN protein to confirm the specificity ofthe recovered virus (FIG. 5). The recovered viruses were designatedrLaSota. To identify the recovered virus, two genetic markers (MluI andSnaBI) were introduced in the full-length cDNA clone. In order to verifythe presence of these markers, RNA from recovered virus was subjected toRT-PCR. DNA fragments encompassing the regions containing the MluI andSnaBI sites were subjected to restriction enzyme digestion with therespective enzymes. Analysis of the restriction enzyme patterns revealedthe presence of both genetic markers in rLaSota, as calculated from thesizes of the bands, while RT-PCR products from wild-type LaSota were notdigested by the enzymes (FIG. 6a).

[0072] Nucleotide sequence analysis of RT-PCR products also confirmedthe presence of the genetic markers. The procedures for RT-PCR anddemonstration of genetic marker are described herein. RNA was isolatedfrom recovered virus by using TRIzol reagent. RT-PCR was performed withprimers P1 (5′ TCCCCTGGTATTTATTCCTGC, positions 5609-5629) and P1 R (5′GTTGGCCACCCAGTCCCCGA, negative sense, positions 7286-7305) to amplify afragment including the introduced Mlul site in the intergenic regionbetween the F and HN genes. Similarly, a fragment containing the SnaBIsite within the HN-L intergenic region was amplified with primers P2 (5′CGCATACAGCAGGCTATCTTATC, positions 7513-7535) and P2R(5′GGGTCATATTCTATACATGGC, negative sense, positions 9739-9759). TheRT-PCR products were then subjected to restriction enzyme digestion, thefirst product with MluI, the second with SnaBI. The restriction patternswere analysed by agarose gel electrophoresis. RT-PCR was also performedto demonstrate the location of the CAT gene insert in the recombinantNDV expressing the CAT gene.

[0073] B. Construction of a Full-Length Plasmid Containing theChloramphenicol Acetyltransferase (CAT) Gene

[0074] For the convenience of inserting CAT into the most 3′-proximallocus, an AscI-SacIl fragment of the full-length cDNA clone wassubcloned into plasmid pGEM-7Z between the XbaI and HindIII sites byusing a specific primer pair with XbaI and HindIII site overhangs. An 18nt insert with a unique PmeI site was then introduced just before the NPORF by the method described previously (Byrappa et al., 1995). To insertthe CAT gene into the PmeI site, the CAT gene ORF was amplified byprimers (5′ gctagtttaaacATGGAGAAAAAAATCACTGGATATACC 3′, positive sense,and 5′ gctagtttaaacttctacccgtgttttttctaatctgcagTTACGCCCCGCCCTGCCACTCATCGC 3′, negative sense; PmeI site and NDV gene start and gene end signalin lower case, CAT-specific sequence in capitals), digested with PmeIand placed into the NP non-coding region in pGEM-7Z (FIG. 4). Cloneswith the CAT gene in the correct orientation were chosen for sequencing.The AscI-SaclI fragment containing the CAT gene was used to replace thecorresponding fragment in pLaSota. Thus, an additional transcriptionalunit, the CAT ORF flanked by NDV gene start and gene end signals, wasinserted into pLaSota. The total number of nucleotides was adjusted byinserting nucleotides after the CAT gene stop codon to maintain the‘rule of six’. The resulting clone was designated pLaSota/CAT.

[0075] The CAT gene ORF, flanked by NDV gene start and gene endsequences, was inserted into the non-coding region of the NP geneimmediately before the NP ORF (FIG. 4). The resulting plasmid wouldencode an antigenome of 15900 nt, obeying the ‘rule of six’ (Peeters etal., 2000). In the recovered virus, the inserted CAT gene would beexpressed as a monocistronic mRNA under the control of the NDVtranscription system. The method for recovery of recombinant NDV was thesame as described above. Plaques produced by rLaSota/CAT wereimmunostained with HN-specific monoclonal antibody and were of a sizeand morphology similar to those produced by rLaSota. The presence of theCAT gene in the genome of recovered virus was verified by RT-PCR witholigonucleotide primers spanning the CAT gene. The size of the RT-PCRproduct from recovered rLaSota was 16 kb, while that from rLaSota/CATwas 23 kb (FIG. 6b). Direct PCR from extracted RNA without RT did notyield any product. Nucleotide sequence analysis of the RT-PCR productconfirmed the presence of the CAT gene in the genome of the recoveredvirus rLaSota/CAT.

[0076] To examine the expression of the CAT protein from rLaSota/CAT,cell lysates from 12 passages, beginning with the third, were tested forCAT activity. For rLaSota/CAT, all passages showed similar CAT enzymeactivity by CAT assay (procedure described below, but data not shown).These results showed that the inserted CAT gene was stable, at least upto passage 12. In Example 2 described below, an NDV-CAT chimerictranscription cassette was inserted between the HN and L genes of thefull-length cDNA of virulent NDV strain Beaudette C and infectiousCAT-expressing recombinant NDV (rBC/CAT) was recovered. In order tocompare the level of expression of the CAT genes from rLaSota/CAT andrBC/CAT, replicate monolayers of DF1 cells were infected with each virusseparately at an m.o.i. of 0.1. Four days after infection, CAT enzymeactivities in the cell lysates were examined (FIG. 7). The resultsshowed that the CAT enzyme activity was about 11-fold higher in cellsinfected with rLaSota/CAT than in cells infected with rBC/CAT.

[0077] The activity of CAT was assayed as described below for analysisof the stability of CAT expression. Chicken embryo fibroblast DF1 cellpellets were lysed by three freeze-thaw cycles and 1% of the lysedpellet from a 25 cm2 flask was analysed by TLC for the ability toacetylate [¹⁴C]chloramphenicol (Amersham Pharmacia). To study thestability of CAT expression by the recombinant virus, a total of 12serial passages were performed at a passage interval of 4 days. At eachpassage, 100 μl of the medium supernatant was used for passing to freshDF1 cells in a 25 cm2 flask. Acetyltrypsin (1 μg/ml) was included in themedium of DF1 cells for cleavage of the F protein of rLaSota andrLaSota/CAT.

[0078] To examine the presence of CAT mRNA and the level of synthesis ofthe immediate downstream NP mRNA, Northern blot hybridization wasperformed with poly(A)+ RNA from cells infected with rLaSota orrLaSota/CAT, each at passage 6. Northern blot hybridization was carriedout as described herein. RNA was isolated from cells infected witheither rLaSota or rLaSota/CAT at an m.o.i. of 1. Total RNA was extractedwith TRIzol reagent and poly(A)+ mRNA was selected by using an mRNAisolation kit (Promega). mRNA samples were subjected to electrophoresison 1-5% agarose gels containing 044 M formaldehyde, transferred tonitrocellulose membrane and used for hybridization with[³²P]CTP-labelled riboprobes. The negative-sense CAT and NP probes wheresynthesized by in vitro transcription of linearized plasmids containingthese genes. Hybridization of the mRNA extracted fromrLaSota/CAT-infected cells with a negative-sense CAT-specific riboprobedetected a single major band of the size predicted for CAT mRNA (FIG.8). Hybridization with a negative-sense riboprobe specific for the NPgene showed a single major band at the size predicted for NP mRNA inboth rLaSota and rLaSota/CAT blots. Densitometry scanning did not show asignificant difference in the level of NP mRNA synthesis between rLaSotaand rLaSota/CAT. This result indicated that insertion of the CAT gene atthe most 3′-proximal locus did not affect mRNA synthesis of theimmediate downstream NP gene significantly.

EXAMPLE 2

[0079] In this working example, an embodiment of the invention in whichthe recombinant NDV containing a gene encoding CAT as the foreign geneinserted between the HN and L genes was prepared.

[0080] A. Construction of a Full-Length NDV cDNA Clone

[0081] A cDNA clone encoding the entire 15,186-nt antigenome of NDVstrain Beaudette C was constructed from 8 cDNA segments that weresynthesized by RT-PCR from NDV Beaudette C derived genomic RNA (FIG. 10;FIG. 11). The oligonucleotide primers used during full-lengthantigenomic cDNA synthesis and RT-PCR are shown in Table 2, in which thecDNA fragments correspond to the DNA fragments in FIG. 11. In Table 2,T7 promoter sequences are marked in italic type, the virus-specificsequences are underlined, and restriction sites are marked in bold type;the partial HDV ribozyme sequence (24-nt) overhang is shown inlowercase; and orientation of the primer sequence is shown for sense (+)and antisense (−). Each cDNA fragment was completely sequenced beforeassembly into the full-length cDNA clone. The leader end was constructedto join a promoter for T7 RNA polymerase. To generate a nearly exact 3′end, the trailer end was constructed to join hepatitis delta virus (HDV)antigenome ribozyme sequence followed by tandem terminators of T7transcription. Two restriction site markers were introduced into theantigenomic cDNA by incorporating the changes into the oligonucleotideprimers used in RT-PCR in order to identify the recombinant virus. AnMlu I site was created in the F-HN intergenic region and the otherunique Age I site was created in the HN-L intergenic region. Cloning ofthese cDNA fragments positioned the NDV cDNA between the T7 promoter andthe hepatitis delta virus ribozyme sequence. The resulting recombinantpBR322 plasmid contained the full-length cDNA encoding the antigenome ofNDV Beaudettel C. To facilitate transcription of the cDNA in the plasmidby T7 RNA polymerase later, three G resides were included before the NDVleader sequence.

[0082] To recover recombinant NDV from the cDNA located in the plasmid,the strategy shown in FIG. 14 was used. HEp2 cells were infected withrecombinant vaccinia virus (MVA/T7) capable of synthesizing T7 RNApolymerase. The HEp2 cells were simultaneously transfected with (1) therecombinant plasmid pBR322 containing the cDNA encoding the antigenomicRNA of NDV Beaudette C, (2) a plasmid containing the NP gene under thecontrol of the T7 promoter, (3) a plasmid containing the P gene underthe control of the T7 promoter, and (4) a plasmid containing the L geneunder the control of the T7 promoter. Three or four days after thetransfection, infectious recombinant NDV was isolated from thesupernatant by one of two ways. The supernatant was either injected intothe allantoic cavities of 9-day-old embryonated eggs or amplifiedfurther in HEp-2 cells and DF1 cells (chicken embryo fibroblast cellline). An antigenomic (+)-sense RNA transcript was produced bytranscription of the NDV full-length antigenomic cDNA.

[0083] B. Construction of Recombinant NDV Having a Foreign Gene InsertedBetween the HN and L Genes

[0084] The recombinant pBR322 plasmid containing the cDNA clone encodingthe entire 15,186-nt antigenome of NDV strain Beaudette C prepared inPart A of Working Example 2 above was used to construct the recombinantNDV having a foreign gene inserted. The gene encoding chloramphenicolacetyltransferase (CAT) was the foreign gene in this example. Thesequence in the NH-L intergenic region of the full-length antigenomecDNA clone of NDV strain Beaudette C was modified to contain a uniqueSna B I restriction site downstream of the Age I restriction site. Theopen reading frame (ORF) encoding the CAT protein was engineered to beflanked by the NDV GS and GE signals. This transcription cassette wasinserted into the HN-L intergenic region of NDV full-length antigenomiccDNA to prepare a recombinant pBR322 containing the full-lengthantigenomic cDNA containing the CAT gene inserted between the HN and Lgenes (see FIG. 12; with a more detailed version shown in FIG. 13). Inthis construct, care was taken over the genome length preference calledthe “rule of six”, i.e. NDV having a preference, but not an absoluterequirement, that the number of nucleotides in the genome is a multipleof six.

[0085] To recover the recombinant NDV containing the CAT gene, thestrategy shown in FIG. 14 was used. The recombinant NDV recoveryprocedures described in Part B of this working example were used exceptthat the HEp2 cells were simultaneously transfected with (1) therecombinant plasmid pBR322 containing NDV antigenomic cDNA having theCAT gene inserted, (2) a plasmid containing the NP gene, (3) a plasmidcontaining the P gene, and (4) a plasmid containing the L gene.

[0086] RT-PCR of the genomic RNA isolated from the recovered virusshowed the presence of the inserted CAT gene. The recovered virusexpressed abundant levels of CAT enzyme. In FIG. 15, lane 1 shows datafrom laboratory Beaudette C strain, and lane 2 shows data fromrecombinant Beaudette C strain containing the CAT gene. Analysis ofmRNAs by Northern blot hybridization showed that the CAT gene wasexpressed as an additional, separate, poly(A) mRNA. CAT expression wasstable for at least 8 passages, indicating that the activity of the CATprotein encoded by NDV remained unimpaired by mutation. There was noappreciable difference either in plaque phenotype or in growth kineticsbetween the virus recovered from the recombinant NDV and wild-typelaboratory NDV strain.

EXAMPLE 3

[0087] Some of the characteristics of the recombinant viruses, rLaSotaand rLaSota/CAT, were determined using the recombinant viruses recoveredfrom transcription of the recombinant cDNA of NDV carrying the CAT geneinserted before the NP gene or between the HN and L genes as obtained inExamples 1 and 2.

[0088] A. Nucleotide Sequences

[0089] The nucleotide sequence of the recombinant cDNA for NDV LaSotaexpressing the CAT gene inserted in front of the NP gene as prepared inExample 1 is shown in Table 3, Parts a-d (labeled as LASO_CAT.TXT). Thenucleotide sequence of the recombinant cDNA for NDV Beaudette Cexpressing the CAT gene inserted between the HN and L genes as preparedin Example 2 is shown in Table 4, Parts a-d (labeled as BC_CAT_.TXT).

[0090] B. Growth Characteristics of the Recombinant Viruses

[0091] The efficiency of replication in tissue culture of rLaSota,rLaSota/CAT and wild-type NDV LaSota was compared in a multiple-stepgrowth cycle. Triplicate monolayers of DF1 cells were infected with eachvirus at an m.o.i. of 0·005 and samples were collected at 8 h intervals.The virus titers of these samples were quantified by plaque assay (FIG.9). Both the kinetics and the magnitude of replication of the threeviruses were very similar. However, the production of rLaSota/CAT wasdelayed slightly compared with rLaSota and wild-type NDV strain LaSota.

[0092] C. Determination of the Intracerebral Pathogenicity Index (ICPI)in 1-Day-Old Chicks

[0093] ICPI was used to determine the virulence of wild-type andrecombinant NDVs in 1-day-old chicks. For each ICPI test, 151-day-oldSPF chicks were used (ten birds for test and five birds for control).The inoculum consisted of fresh, infective allantoic fluid with an HAtiter >24 (1:16) for the test birds and allantoic fluid from uninfectedembryonated chicken eggs for control birds. Both inocula were diluted1:10 in sterile PBS. Each bird was inoculated intracerebrally with 005ml inoculum. The birds were observed for clinical signs and mortalityevery 24 h for a period of 8 days. The scoring and determination of ICPIwere done according to the method described by Alexander (1997).

[0094] In order to compare the pathogenicity of rLaSota, rLaSota/CAT andwild-type NDV strain LaSota, ICPI tests in 1-day-old chicks wereperformed by scoring clinical signs and mortality. The most virulent NDVstrains give indices close to 2.0, while avirulent viruses give valuesclose to 0. In our experiment, the results of ICPI were 027 forwild-type NDV LaSota, 029 for rLaSota and 024 for rLaSota/CAT. Theseresults show that the recombinant viruses were similar in virulence towild-type NDV strain LaSota.

[0095] The results described here show that attenuated NDV can be usedas a vaccine vector to express a foreign gene. Development ofrecombinant NDV as a vaccine vector has several applications. Severalforeign genes can be inserted and expressed in the same virus to obtainsimultaneous immune responses to the expressed antigens in inoculatedanimals. For example, a single recombinant NDV could be generated thatexpressed the immunogenic proteins of multiple avian pathogens.Alternatively, several NDVs, each expressing various heterologousantigens, could be administered as a multivalent vaccine. A furtherextension would be to use NDV vectors in non-avian species, where NDV iscapable of undergoing incomplete replication to the extent necessary toexpress inserted genes. Thus, development of NDV as a vector shouldprove to be useful against avian and non-avian diseases for whichsuitable vaccines are not currently available.

EXAMPLE 4

[0096] A recombinant cDNA to the genome of NDV strain Beaudette C havinga foreign gene, a gene encoding green fluorescent protein (GFP),inserted between P and M genes was prepared by inserting the GFP geneinto the noncoding region of P gene after the P gene ORF and stop codon,but before the P gene GE signal (see FIG. 16). To allow the ORF of theGFP gene to be inserted into the noncoding region of the P gene, a XbaIsite was created via mutation of a TCTCGC segment (nucleotide positions3182-3187) in the P gene noncoding region after a stop codon forming aTCTAGA segment. The ORF of the GFP gene was preceded by a cDNA segment,TTAGAAAAAA, for a NDV gene end signal followed by a cDNA segment,ACGGGTAGM, for a NDV gene start signal. Transcription of a plasmidcontaining the recombinant cDNA to the NDV Beaudette C genome carryingthe GFP gene inserted into the noncoding region of the P gene resultedin a recombinant NDV which was found to be able to express GFP.

REFERENCES

[0097] Alexander, D. J. (1997). Newcastle disease and other avianParamyxoviridae infections. In Diseases of Poultry, 10^(th) edition, pp.541-569. Edited by B. W. Calnek, Iowa State University Press, Ames,Iowa.

[0098] Byrappa, S., Gavin, D. K. & Gupta, K. C. (1995). A highlyefficient procedure for site-specific mutagenesis of full-lengthplasmids using Vent DNA polymerase. Genome Research 5, 404-407.

[0099] Conzelmann, K.-K. (1996). Genetic manipulation of non-segmentednegative-strand RNA viruses. Journal of General Virology 77, 381-389.

[0100] de Leeuw, O. & Peeters, B. (1999). Complete nucleotide sequenceof Newcastle disease virus: evidence for the existence of a new genuswithin the subfamily Paramyxovirinae. Journal of General Virology 80,131-136.

[0101] Kingsbury, D. W. (1966). Newcastle disease virus. I. Isolationand preliminary characterization of RNA from virus particles. Journal ofMolecular Biology 18, 195-203.

[0102] Krishnamurthy, S. & Samal, S. K. (1998). Nucleotide sequences ofthe trailer, nucleocapsid protein gene and intergenic regions ofNewcastle disease virus strain Beaudette C and completion of the entiregenome sequence. Journal of General Virology 79, 2419-2424.

[0103] Peeters, B. P., de Leeuw, O. S., Koch, G. & Gielkens, A. L.(1999). Rescue of Newcastle disease virus from cloned cDNA: evidencethat cleavability of the fusion protein is a major determinant forvirulence. Journal of Virology 73, 5001-5009.

[0104] Phillips, R. J., Samson, A. R. & Emmerson, P. T. (1998).Nucleotide sequence of the 5′-terminus of Newcastle disease virus andassembly of the complete genomic sequence: agreement with the ‘rule ofsix’. Archives of Virology 143, 1993-2002.

[0105] Römer-Oberdorfer, A., Mundt, E., Mebatsion, T., Buchholz, U. J. &Mettenleiter, T. C. (1999). Generation of recombinant lentogenicNewcastle disease virus from cDNA. Journal of General Virology 80,2987-2995.

[0106] Steward, M., Vipond, I. B., Millar, N. S. & Emmerson, P. T.(1993). RNA editing in Newcastle disease virus. Journal of GeneralVirology 74, 2539-2547. TABLE 1 Oligonucleotide primers used for RT-PCRand assembly of full-length cDNA cDNA Primer Order of fragment SenseAntisense cloning I 5′ CTGAGGCGCGCCTAATACGACTCACTATAGG 5′ GTTTCCGCGGCTGGGTTGACTCCCCT 3′ 4 ACCAAACAGAGAATCCGTGAGTTAG 3′ II 5′ GGTGCCGCGGAAACAGCCAGG 3′ 5′ GAGCTGCGGCCGCTGTTATTTG 3′ 6 III5′ AACAGCGGCCGCAGCTCTGAT 3′ 5′ TACAAC GC GTAGTTTTTTCTTAACTC 3′ 7 IV5′ AACTAC GC GTTGTAGATGACCAAAG 3′ 5′ GCACTACGTA TTTTGCCTTGTATCTC 3′ 5 V5′ CAAAATACGTA ATCGTAAATAATACGGGT 5′ TTCA GCTTAGCGAAGATCCGTCCATTAACT 3′3 AGGACATG 3′ VI 5′ TTCA GCTAAGCTGACAAAGAAGTTAAGG5′ GTCTAGGCCTCTTACTCTCAGGTAATAG 3′ 1 AACTG 3′ VII 5′ TCAGAGGCCTAGACAATATTGTCT 3′ 5′ GATCCGGACCGcgaggaggtggagatgccatgccg 2ACCAAACAAAGATTTGGTGAATGACGAG 3′

[0107] TABLE 2 Oligonucleotide Primers Used during Full-Length cDNASynthesis and RT-PCR cDNA Order of fragments Primers cloning I+ 5′ACTGGGGCGCGCTAATACGACTCACTATAGGACCAAACAGAGAATCCGTAAGTTAG3′ 8− 5′AGACC CGCGGCTGGGTTGACTTCCCTG3′ II + 5′AGAC CCGCGGAAACAGCCAGG3′ 7− 5′GCAG GGGCCCATCTTGCACCTAGAA3′ III + 5′ACAG GGGCCCCAGACCTTCTACCAA3′ 6− 5′ATCG AC GCGTAGTTTTTTCTAAACTCTC3′ IV + 5′ATCGACGCGTTGTAGATGACCAAAG3′5 − 5′GCACACCGGTAGCTGTTTTGCCTTGTATC3′ V+ 5′GCACACCGGTAAATAGTACGGGTAGGACATG3′ 2 − 5′TTCAGCTTAGCGAAGATCCGTCCATTAAGT3′ VI + 5′TTCA GCTAAGCTGACAAAGAAGTTAAGGAACTG3′4 − 5′AAGC CTTAAGAACAATGTTTGGGCTTGCAAC3′ VII + 5′AAGCCTTAAGAAACATACGCAAAGAGTCCT3′ 3 − 5′TCAG AGGCCTTCTTACTCTCAGATAATAGAG3′VIII − 5′TCAG AGGCCTTCTTACTCTCAGATAATAGAG3′ 1  5′ATGCCGGACCGcgaggaggtggagatgccatgccgACCCACCAAACAAAGATTTGGTGAATAACAAG3′

[0108] TABLE 3 LASO_CAT.TXT (Part a)ACCAAACAGAGAATCCGTGAGTTACGATAAAAGGCGAAAGAGCAATTGAAGTCGCACGGGTAGAAGGTGTGAATCTCGAGTGCGAGCCCGAAGCACAAACTCGAGAAAGCCTTCTGCCAACGTTTAAACATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATACTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAACTGCAGATTAGAAAAAACACGGGTAGAAGTTTAAACTAGGTGCAAGATGTCTTCCGTATTTGATGAGTACGAACAGCTCCTCGCGGCTCAGACTCGCCCCAATGGAGCTCATGGAGGGGGAGAAAAAGGGAGTACCTTAAAAGTAGACGTCCCGGTATTCACTCTTAACAGTGATGACCCAGAAGATAGATGGAGCTTTGTGGTATTCTGCCTCCGGATTGCTGTTAGCGAAGATGCCAACAAACCACTCAGGCAAGGTGCTCTCATATCTCTTTTATGCTCCCACTCACAGGTAATGAGGAACCATGTTGCCCTTGCAGGGAAACAGAATGAAGCCACATTGGCCGTGCTTGAGATTGATGGCTTTGCCAACGGCACGCCCCAGTTCAACAATAGGAGTGGAGTGTCTGAAGAGAGAGCACAGAGATTTGCGATGATAGCAGGATCTCTCCCTCGGGCATGCAGCAACGGAACCCCGTTCGTCACAGCCGGGGCCGAAGATGATGCACCAGAAGACATCACCGATACCCTGGAGAGGATCCTCTCTATCCAGGCTCAAGTATGGGTCACAGTAGCAAAAGCCATGACTGCGTATGAGACTGCAGATGAGTCGGAAACAAGGCGAATCAATAAGTATATGCAGCAAGGCAGGGTCCAAAAGAAATACATCCTCTACCCCGTATGCAGGAGCACAATCCAACTCACGATCAGACAGTCTCTTGCAGTCCGCATCTTTTTGGTTAGCGAGCTCAAGAGAGGCCGCAACACGGCAGGTGGTACCTCTACTTATTATAACCTGGTAGGGGACGTAGACTCATACATCAGGAATACCGGGCTTACTGCATTCTTCTTGACACTCAAGTACGGAATCAACACCAAGACATCAGCCCTTGCACTTAGTAGCCTCTCAGGCGACATCCAGAAGATGAAGCAGCTCATGCGTTTGTATCGGATGAAAGGAGATAATGCGCCGTACATGACATTACTTGGTGATAGTGACCAGATGAGCTTTGCGCCTGCCGAGTATGCACAACTTTACTCCCTTGCCATGGGTATGGCATCAGTCCTAGATAAAGGTACTGGGAAATACCAATTTGCCAGGGACTTTATGAGCACATCATTCTGGAGACTTGGAGTAGAGTACGCTCAGGCTCAGGGAAGTAGCATTAACGAGGATATGGCTGCCGAGCTAAAGCTAACCCCAGCAGCAAGGAGGGGCCTGGCAGCTGCTGCCCAACGGGTCTCCGAGGAGACCAGCAGCATAGACATGCCTACTCAACAAGTCGGAGTCCTCACTGGGCTTAGCGAGGGGGGGTCCCAAGCTCTACAAGGCGGATCGAATAGATCGCAAGGGCAACCAGAAGCCGGGGATGGGGAGACCCAATTCCTGGATCTGATGAGAGCGGTAGCAAATAGCATGAGGGAGGCGCCAAACTCTGCACAGGGCACTCCCCAATCGGGGCCTCCCCCAACTCCTGGGCCATCCCAAGATAACGACACCGACTGGGGGTATTGATGGACAAAACCCAGCCTGCTTCCACAAAAACATCCCAATGCCCTCACCCGTAGTCGACCCCTCGATTTGCGGCTCTATATGACCACACCCTCAAACAAACATCCCCCTCTTTCCTCCCTCCCCCTGCTGTACAACTCCGCACGCCCTAGATACCACAGGCACAATGCGGCTCACTAACAATCAAAACAGAGCCGAGGGAATTAGAAAAAAGTACGGGTAGAAGAGGGATATTCAGAGATCAGGGCAAGTCTCCCGAGTCTCTGCTCTCTCCTCTACCTGATAGACCAGGACAAACATGGCCACCTTTACAGATGCAGAGATCGACGAGCTATTTGAGACAAGTGGAACTGTCATTGACAACATAATTACAGCCCAGGGTAAACCAGCAGAGACTGTTGGAAGGAGTGCAATCCCACAAGGCAAGACCAAGGTGCTGAGCGCAGCATGGGAGAAGCATGGGAGCATCCAGCCACCGGCCAGTCAAGACAACCCCGATCGACAGGACAGATCTGACAAACAACCATCCACACCCGAGCAAACGACCCCGCATGACAGCCCGCCGGCCACATCCGCCGACCAGCCCCCCACCCAGGCCACAGACGAAGCCGTCGACACACAGCTCAGGACCGGAGCAAGCAACTCTCTGCTGTTGATGCTTGACAAGCTCAGCAATAAATCGTCCAATGCTAAAAAGGGCCCATGGTCGAGCCCCCAAGAGGGGAATCACCAACGTCCGACTCAACAGCAGGGGAGTCAACCCAGCCGCGGAAACAGTCAGGAAAGACCGCAGAACCAAGTCAAGGCCGCCCCTGGAAACCAGGGCACAGACGTGAACACAGCATATCATGGACAATGGGAGGAGTCACAACTATCAGCTGGTGCAACCCCTCATGCTCTCCGATCAAGGCAGAGCCAAGACAATACCCTTGTATCTGCGGATCATGTCCAGCCACCTGTAGACTTTGTGCAAGCGATGATGTCTATGATGGAGGCGATATCACAGAGAGTAAGTAAGGTCGACTATCAGCTAGATCTTGTCTTGAAACAGACATCCTCCATCCCTATGATGCGGTCCGAAATCCAACAGCTGAAAACATCTGTTGCAGTCATGGAAGCCAACTTGGGAATGATGAAGATTCTGGATCCCGGTTGTGCCAACATTTCATCTCTGAGTGATCTACGGGCAGTTGCCCGATCTCACCCGGTTTTAGTTTCAGGCCCTGGAGACCCCTCTCCCTATGTGACACAAGGAGGCGAAATGGCACTTAATAAACTTTCGCAACCAGTGCCACATCCATCTGAATTGATTAAACCCGCCACTGCATGCGGGCCTGATATAGGAGTGGAAAAGGACACTGTCCGTGCATTGATCATGTCACGCCCAATGCACCCGAGTTCTTCAGCCAAGCTCCTAAGCAAGTTAGATGCAGCCGGGTCGATCGAGGAAATCAGGAAAATCAAGCGCCTTGCTCTAAATGGCTAATTACTACTGCCACACGTAGCGGGTCCCTGTCCACTCGGCATCACACGGAATCTGCACCGAGTTCCCCCCCGCAGACCCAAGGTCCAACTCTCCAAGCGGCAATCCTCTCTCGCTTCCTCAGCCCCACTGAATGGTCGCGTAACCGTAATTAATCTAGCTACATTTAAGATTAAGAAAAAATACGGGTAGAATTGGAGTGCCCCAATTGTGCCAAGATGGACTCATCTAGGACAATTGGGCTGTACTTTGATTCTGCCCATTCTTCTAGCAACCTGTTAGCATTTCCGATCGTCCTACAAGGCACAGGAGATGGGAAGAAGCAAATCGCCCCGCAATATAGGATCCAGCGCCTTGACTTGTGGACTGATAGTAAGGAGGACTCAGTATTCATCACCACCTATGGATTCATCTTTCAAGTTGGGAATGAAGAAGCCACTGTCGGCATGATCGATGATAAACCCAAGCGCGAGTTACTTTCCGCTGCGATGCTCTGCCTAGGAAGCGTCCCAAATACCGGAGACCTTATTGAGCTGGCAAGGGCCTGTCTCACTATGATAGTCACATGCAAGAAGAGTGCAACTAATACTGAGAGAATGGTTTTCTCAGTAGTGCAGGCACCCCAAGTGCTGCAAAGCTGTAGGGTTGTGGCAAACAAATACTCATCAGTGAATGCAGTCAAGCACGTGAAAGCGCCAGAGAAGATTCCCGGGAGTGGAACCCTAGAATACAAGGTGAACTTTGTCTCCTTGACTGTGGTACCGAAGAAGGATGTCTACAAGATCCCTGCTGCAGTATTGAAGGTTTCTGGCTCGAGTCTGTACAATCTTGCGCTCAATGTCACTATTAATGTGGAGGTAGACCCGAGGAGTCCTTTGGTTAAATCTCTGTCTAAGTCTGACAGCGGATACTATGCTAACCTCTTCTTGCATATTGGACTTATGACCACCGTAGATAGGAAGGGGAAGAAAGTGACATTTGACAAGCTGGAAAAGAAAATAAGGAGCCTTGATCTATCTGTCGGGCTCAGTGATGTGCTCGGGCCTTCCGTGTTGGTAAAAGCAAGAGGTGCACGGACTAAGCTTTTGGCACCTTTCTTCTCTAGCAGTGGGACAGCCTGCTATCCCATAGCAAATGCTTCTCCTCAGGTGGCCAAGATACTCTGGAGTCAAACCGCGTGCCTGCGGAGCGTTAAAATCATTATCCAAGCAGGTACCCAACGCGCTGTCGCAGTGACCGCCGACCACGAGGTTACCTCTACTAAGCTGGAGAAGGGGCACACCCTTGCCAAATACAATCCTTTTAAGAAATAAGCTGCGTCTCTGAGATTGCGCTCCGCCCACTCACCCAGATCATCATGACACAAAAAACTAATCTGTCTTGATTATTTACAGTTAGTTTACCTGTCTATCAAGTTAGAAAAAACACGGGTAGAAGATTCTGGATCCCGGTTGGCGCCCTCCAGGTGCAAGATGGGCTCCAGACCTTCTACCAAGAACCCAGCACC(Part b)TATGATGCTGACTATCCGGGTTGCGCTGGTACTGAGTTGCATCTGTCCGGCAAACTCCATTGATGGCAGGCCTCTTGCAGCTGCAGGAATTGTGGTTACAGGAGACAAAGCCGTCAACATATACACCTCATCCCAGACAGGATCAATCATAGTTAAGCTCCTCCCGAATCTGCCCAAGGATAAGGAGGCATGTGCGAAAGCCCCCTTGGATGCATACAACAGGACATTGACCACTTTGCTCACCCCCCTTGGTGACTCTATCCGTAGGATACAAGAGTCTGTGACTACATCTGGAGGGGGGAGACAGGGGCGCCTTATAGGCGCCATTATTGGCGGTGTGGCTCTTGGGGTTGCAACTGCCGCACAAATAACAGCGGCCGCAGCTCTGATACAAGCCAAACAAAATGCTGCCAACATCCTCCGACTTAAAGAGAGCATTGCCGCAACCAATGAGGCTGTGCATGAGGTCACTGACGGATTATCGCAACTAGCAGTGGCAGTTGGGAAGATGCAGCAGTTTGTTAATGACCAATTTAATAAAACAGCTCAGGAATTAGACTGCATCAAAATTGCACAGCAAGTTGGTGTAGAGCTCAACCTGTACCTAACCGAATTGACTACAGTATTCGGACCACAAATCACTTCACCTGCTTTAAACAAGCTGACTATTCAGGCACTTTACAATCTAGCTGGTGGAAATATGGATTACTTATTGACTAAGTTAGGTGTAGGGAACAATCAACTCAGCTCATTAATCGGTAGCGGCTTAATCACCGGTAACCCTATTCTATACGACTCACAGACTCAACTCTTGGGTATACAGGTAACTCTACCTTCAGTCGGGAACCTAAATAATATGCGTGCCACCTACTTGGAAACCTTATCCGTAAGCACAACCAGGGGATTTGCCTCGGCACTTGTCCCAAAAGTGGTGACACAGGTCGGTTCTGTGATAGAAGAACTTGACACCTCATACTGTATAGAAACTGACTTAGATTTATATTGTACAAGAATAGTAACGTTCCCTATGTCCCCTGGTATTTATTCCTGCTTGAGCGGCAATACGTCGGCCTGTATGTACTCAAAGACCGAAGGCGCACTTACTACACCATACATGACTATCAAAGGTTCAGTCATCGCCAACTGCAAGATGACAACATGTAGATGTGTAAACCCCCCGGGTATCATATCGCAAAACTATGGAGAAGCCGTGTCTCTAATAGATAAACAATCATGCAATGTTTTATCCTTAGGCGGGATAACTTTAAGGCTCAGTGGGGAATTCGATGTAACTTATCAGAAGAATATCTCAATACAAGATTCTCAAGTAATAATAACAGGCAATCTTGATATCTCAACTGAGCTTGGGAATGTCAACAACTCGATCAGTAATGCTTTGAATAAGTTAGAGGAAAGCAACAGAAAACTAGACAAAGTCAATGTCAAACTGACCAGCACATCTGCTCTCATTACCTATATCGTTTTGACTATCATATCTCTTGTTTTTGGTATACTTAGCCTGATTCTAGCATGCTACCTAATGTACAAGCAAAAGGCGCAACAAAAGACCTTATTATGGCTTGGGAATAATACCCTAGATCAGATGAGAGCCACTACAAAAATGTGAACACAGATGAGGAACGAAGGTTTCCCTAATAGTAATTTGTGTGAAAGTTCTGGTAGTCTGTCAGTTCAGAGAGTTAAGAAAAAACTACGCGTTGTAGATGACCAAAGGACGATATACGGGTAGAACGGTAAGAGAGGCCGCCCCTCAATTGCGAGCCAGGCTTCACAACCTCCGTTCTACCGCTTCACCGACAACAGTCCTCAATCATGGACCGCGCCGTTAGCCAAGTTGCGTTAGAGAATGATGAAAGAGAGGCAAAAAATACATGGCGCTTGATATTCCGGATTGCAATCTTATTCTTAACAGTAGTGACCTTGGCTATATCTGTAGCCTCCCTTTTATATAGCATGGGGGCTAGCACACCTAGCGATCTTGTAGGCATACCGACTAGGATTTCCAGGGCAGAAGAAAAGATTACATCTACACTTGGTTCCAATCAAGATGTAGTAGATAGGATATATAAGCAAGTGGCCCTTGAGTCTCCGTTGGCATTGTTAAAAACTGAGACCACAATTATGAACGCAATAACATCTCTCTCTTATCAGATTAATGGAGCTGCAAACAACAGTGGGTGGGGGGCACCTATCCATGACCCAGATTATATAGGGGGGATAGGCAAAGAACTCATTGTAGATGATGCTAGTGATGTCACATCATTCTATCCCTCTGCATTTCAAGAACATCTGAATTTTATCCCGGCGCCTACTACAGGATCAGGTTGCACTCGAATACCCTCATTTGACATGAGTGCTACCCATTACTGCTACACCCATAATGTAATATTGTCTGGATGCAGAGATCACTCACATTCATATCAGTATTTAGCACTTGGTGTGCTCCGGACATCTGCAACAGGGAGGGTATTCTTTTCTACTCTGCGTTCCATCAACCTGGACGACACCCAAAATCGGAAGTCTTGCAGTGTGAGTGCAACTCCCCT6GGTTGTGATATGCTGTGCTCGAAAGTCACGGAGACAGAGGAAGAAGATTATAACTCAGCTGTCCCTACGCGGATGGTACATGGGAGGTTAGGGTTCGACGGCCAGTACCACGAAAAGGACCTAGATGTCACAACATTATTCGGGGACTGGGTGGCCAACTACCCAGGAGTAGGGGGTGGATCTTTTATTGACAGCCGCGTATGGTTCTCAGTCTACGGAGGGTTAAAACCCAATTCACCCAGTGACACTGTACAGGAAGGGAAATATGTGATATACAAGCGATACAATGACACATGCCCAGATGAGCAAGACTACCAGATTCGAATGGCCAAGTCTTCGTATAAGCCTGGACGGTTTGGTGGGAAACGCATACAGCAGGCTATCTTATCTATCAAGGTGTCAACATCCTTAGGCGAAGACCCGGTACTGACTGTACCGCCCAACACAGTCAVACTCATGGGGGCCGAAGGCAGAATTCTCACAGTAGGGACATCTCATTTCTTGTATCAACGAGGGTCATCATACTTCTCTCCCGCGTTATTATATCCTATGACAGTCAGCAACAAAACAGCCACTCTTCATAGTCCTTATACATTCAATGCCTTCACTCGGCCAGGTAGTATCCCTTGCCAGGCTTCAGCAAGATGCCCCAACTCGTGTGTTACTGGAGTCTATACAGATCCATATCCCCTAATCTTCTATAGAAACCACACCTTGCGAGGGGTATTCGGGACAATGCTTGATGGTGTACAAGCAAGACTTAACCCTGCGTCTGCAGTATTCGATAGCACATCCCGCAGTCGCATTACTCGAGTGAGTTCAAGCAGTACCAAAGCAGCATACACAACATCAACTTGTTTTAAAGTGGTCAAGACTAATAAGACCTATTGTCTCAGCATTGCTGAAATATCTAATACTCTCTTCGGAGAATTCAGAATCGTCCCGTTACTAGTTGAGATCCTCAAAGATGACGGGGTTAGAGAAGCCAGGTCTGGCTAGTTGAGTCAATTATAAAGGAGTTGGAAAGATGGCATTGTATCACCTATCTTCTGCGACATCAAGAATCAAACCGAATGCCGGCGCGTGCTCGAATTCCATGTTGCCAGTTGACCACAATCAGCCAGTGCTCATGCGATCAGATTAAGCCTTGTCATTAATCTCTTGATTAAGAAAAAATGTAAGTGGCAATGAGATACAAGGCAAAATACGTACCGGTAAATAATACGGGTAGGACATGGCGAGCTCCGGTCCTGAAAGGGCAGAGCATCAGATTATCCTACCAGAGCCACACCTGTCTTCACCATTGGTCAAGCACAAACTACTCTATTACTGGAAATTAACTGGGCTACCGCTTCCTGATGAATGTGACTTCGACCACCTCATTCTCAGCCGACAATGGAAAAAAATACTTGAATCGGCCTCTCCTGATACTGAGAGAATGATAAAACTCGGAAGGGCAGTACACCAAACTCTTAACCACAATTCCAGAATAACCGGAGTGCTCCACCCCAGGTGTTTAGAACAACTGGCTAATATTGAGGTCCCAGATTCAACCAACAAATTTCGGAAGATTGAGAAGAAGATCCAAATTCACAACACGAGATATGGAGAACTGTTCACAAGGCTGTGTACGCATATAGAGAAGAAACTGCTGGGGTCATCTTGGTCTAACAATGTCCCCCGGTCAGAGGAGTTCAGCAGCATTCGTACGGATCCGGCATTCTGGTTTCACTCAAAATGGTCCACAGCCAAGTTTGCATGGCTCCATATAAAACAGATCCAGAGGCATCTGATGGTGGCAGCTAAGACAAGGTCTGCGGCCAACAAATTGGTGATGCTAACCCATAAGGTAGGCCAAGTCTTTGTCACTCCTGAACTTGTCGTTGTGACGCATACGAATGAGAACAAGTTCACATGTCTTACCCAGGAACTTGTATTGATGTATGCAGATATGATGGAGGGCAGAGATATGGTCAACATAATATCAACCACGGCGGTGCATCTCAGAAGCTTATCAGAGAAAATTGATGACATTTTGCGGTTAATAGACGCTCTGGCAAAAGACTTGGGTAATCAAGTCTACGATGTTGTATCACTAATGGAGGGATTTGCATACGGAGCTGTCCAGCTACTCGAGCCGTCAGGTACATTTGCAGGAGATTTCTTCGCATTCAACCTGCAGGAGCTTAAAGACATTCTAATTGGCCTCCTCCCCAATGATATAGCAGAATCCGTGACTCATGCAATCGCTACTGTATTCTCTGGTTTAGAACAGAATCAAGCAGCTGAGATGTTGTGTCTGTTGCGTCTGTGGGGTCACCCACTGCTTGAGTCCCGTATTGCAGCAAAGGCAGTCAGGAGCCAAATGTGCGCACCGAAAATGGTAGACTTTGATATGATCCTTCAGGTACTGTCTTTCTTCAAGGGAACAATCATCAACGGGTACAGAAAGAAGAATGCAGGTGTGTGGCCGCGAGTCAAAGTGGATACAATATATGGGAAGGTCATTGGGCAACTACATGCAGATTCAGCAGAGATTTCACACGATATCATGTTGAGAGAGTATAAGAGTTTATCTGCACTTGAATTTGAGCCATGTATAGAATATGACCCTGTCACCAACCTGAGCATGTTCCTAAAAGACAAGGCAATCGCACACCCCAACGATAATTGGCTTGCCTCGTTTAGGCGGAACCTTCTCTCCGAAGACCAGAAGAAACATGT(Part c)AAAAGAAGCAACTTCGACTAATCGCCTCTTGATAGAGTTTTTAGAGTCAAATGATTTTGATCCATATAAAGAGATGGAATATCTGACGACCCTTGAGTACCTTAGAGATGACAATGTGGCAGTATCATACTCGCTCAAGGAGAAGGAAGTGAAAGTTAATGGACGGATCTTCGCTAAGCTGACAAAGAAGTTAAGGAACTGTCAGGTGATGGCGGAAGGGATCCTAGCCGATCAGATTGCACCTTTCTTTCAGGGAAATGGAGTCATTCAGGATAGCATATCCTTGACCAAGAGTATGCTAGCGATGAGTCAACTGTCITTTAACAGCAATAAGAAACGTATCACTGACTGTAAAGAAAGAGTATCTTCAAACCGCAATCATGATCCGAAAAGCAAGAACCGTCGGAGAGTTGCAACCTTCATAACAACTGACCTGCAAAAGTACTGTCTTAATTGGAGATATCAGACAATCAAATTGTTCGCTCATGCCATCAATCACTTGATGGGCCTACCTCACTTCTTCGAATGGATTCACCTAAGACTGATGGACACTACGATGTTCGTAGGAGACCCTTTCAATCCTCCAAGTGACCCTACTGACTGTGACCTCTCAAGAGTCCCTAATGATGACATATATATTGTCAGTGCCAGAGGGGGTATCGAAGGATTATGCCAGAAGCTATGGACAATGATCTCAATTGCTGCAATCCAACTTGCTGCAGCTAGATCGCATTGTCGTGTTGCCTGTATGGTACAGGGTGATAATCAAGTAAIAGCAGTAACGAGAGAGGTAAGATCAGACGACTCTCCGGAGATGGTGTTGACACAGTTGCATCAAGCCAGTGATAATTTCTTCAAGGAATTAATTCATGTCAATCATTTGATTGGCCATAATTTGAAGGATCGTGAAACCATCAGGTCAGACACATTCTTCATATACAGCAAACGAATCTTCAAAGATGGAGCAATCCTCAGTCAAGTCCTCAAAAATTCATCTAAATTAGTGCTAGTGTCAGGTGATCTCAGTGAAAACACCGTAATGTCCTGTGCCAACATTGCCTCTACTGTAGCACGGCTATGCGAGAACGGGCTTCCCAAAGACTTCTGTTACTATTTAAACTATATAATGAGTTGTGTGCAGACATACTTTGACTCTGAGTTCTCCATCACCAACAATTCGCACCCCGATCTTAATCAGTCGTGGATTGAGGACATCTCTTTTGTGCACTCATATGTTCTGACICCTGCCCAATTAGGGGGACTGAGTAACCTTCAATACTCAAGGCTCTACACTAGAAATATCGGTGACCCGGGGACTACTGCTTTTGCAGAGATCAAGCGACTAGAAGCAGTGGGATTACTGAGTCCTAACATTATGACTAATATCTTAACTAGGCCGCCTGGGAATGGAGATTGGGCCAGTCTGTGCAACGACCCATACTCTTTCAATTTTGAGACTGTTGCAAGCCCAAATATTGTTCTTAAGAAACATACGCAAAGAGTCCTATTTGAAACTTGTTCAAATCCCTTATTGTCTGGAGTGCACACAGAGGATAATGAGGCAGAAGAGAAGGCATTGGCTGAATTCTTGCTTAATCAAGAGGTGATTCATCCCCGCGTTGCGCATGCCATCATGGAGGCAAGCTCTGTAGGTAGGAGAAAGCAAATTCAAGGGCTTGTTGACACAACAAACACCGTAATTAAGATTGCGCTTACTAGGAGGCCATTAGGCATCAAGAGGCTGATGCGGATAGTCAATTATTCTAGCATGCATGCAATGCTGTTTAGAGACGATGTTTTTTCCTCCAGTAGATCCAACCACCCCTTAGTCTCTTCTAATATGTGTTCTCTGACACTGGCAGACTATGCACGGAATAGAAGCTGGTCACCTTTGACGGGAGGCAGGAAAATACTGGGTGTATCTAATCCTGATACGATAGAACTCGTAGAGGGTGAGATTCTTAGTGTAAGCGGAGGGTGTACAAGATGTGACAGCGGAGATGAACAATTTACTTGGTTCCATCTTCCAAGCAATATAGAATTGACCGATGACACCAGCAAGAATCCTCCGATGAGGGTACCATATCTCGGGTCAAAGACACAGGAGAGGAGAGCTGCCTCACTTGCAAAAATAGCTCATATGTCGCCACATGTAAAGGCTGCCCTAAGGGCATCATCCGTGTTGATCTGGGCTTATGGGGATAATGAAGTAAATTGGACTGCTGCTCTTACGATTGCAAAATCTCGGTGTAATGTAAACTTAGAGTATCTTCGGTTACTGTCCCCTTTACCCACGGCTGGGAATCTTCAACATAGACTAGATGATGGTATAACTCAGATGACATTCACCCCTGCATCTCTCTACAGGTGTCACCTTACATTCACATATCCAATGATTCTCAAAGGCTGTTCACTGAAGAAGGAGTCAAAGAGGGGAATGTGGTTTACCAACAGAGTCATGCTCTTGGGTTTATCTCTAATCGAATCGATCITTCCAATGACAACAACCAGGACATATGATGAGATCACACTGCACCTACATAGTAAATTTAGTTGCTGTATCAGAGAAGCACCTGTTGCGGTTCCTTTCGAGCTACTTGGGGTGGTACCGGAACTGAGGACAGTGACCTCAAATAAGTTTATGTAIGATCCTAGCCCTGTATCGGAGGGAGACTTTGCGAGACTTGACTTAGCTATCTTCAAGAGTTATGAGCTTAATCTGGAGTCATATCCCACGATAGAGCTAATGAACATTCTTTCAATATCCAGCGGGAAGTTGATTGGCCAGTCTGTGGTTTCTTATGATGAAGATACCTCCATAAAGAATGACGCCATAATAGTGTATGACAATACCCGAAATTGGATCAGTGAAGCTCAGAATTCAGATGTGGTCCGCCTATTTGAATATGCAGCACTTGAAGTGCTCCTCGACTGTTCTTACCAACTCTATTACCTGAGAGTAAGAGGCCTAGACAATATTGTCTTATATATGGGTGATTTATACAAGAATATGCCAGGAATTCTACTTTCCAACATTGCAGCTACAATATCTCATCCCGTCATTCATTCAAGGTTACATGCAGTGGGCCTGGTCAACCATGACGGATCACACCAACTTGCAGATACGGATTTTATCGAAATGTCTGCAAAACTATTAGTATCTTGCACCCGACGTGTGATCTCCGGCTTATATTCAGGAAATAAGTATGATCTGCTGTTCCCATCTGTCTTAGATGATAACCTGAATGAGAAGATGCTTCAGCTGATATCCCGGTTATGCTGTCTGTACACGGTACTCTTTGCTACAACAAGAGAAATCCCGAAAATAAGAGGCTTAACTGCAGAAGAGAAATGTTCAATACTCACTGAGTATTTACTGTCGGATGCTGTGAAACCATTACTTAGCCCCGATCAAGTGAGCTCTATCATGTCTCCTAACATAATTACATTCCCAGCTAATCTGTACTACATGTCTCGGAAGAGCCTCAATTTGATCAGGGAAAGGGAGGACAGGGATACTATCCTGGCGTTGTTGTTCCCCCAAGAGCCATTATTAGAGTTCCCTTCTGTGCAAGATATTGGTGCTCGAGTGAAAGATCCATTCACCCGACAACCTGCGGCATTTTTGCAAGAGTTAGATTTGAGTGCTCCAGCAAGGTATGACGCATTCACACTTAGTCAGATTCATCCTGAACTCACATCTCCAAATCCGGAGGAAGACTACTTAGTACGATACTTGTTCAGAGGGATAGGGACTGCATCTTCCTCTTGGTATAAGGCATCTCATCTCCTTTCTGTACCCGAGGTAAGATGTGCAAGACACGGGAACTCCTTATACTTAGCTGAAGGGAGCGGAGCCATCATGAGTCTTCTCGAACTGCATGTACCACATGAAACTATCTATTACAATACGCTCTTTTCAAATGAGATGAACCCCCCGCAACGACATTTCGGGCCGACCCCAACTCAGTTTTTGAATTCGGTTGTTTATAGGAATCTACAGGCGGAGGTAACATGCAAAGATGGATTTGTCCAAGAGTTCCGTCCATTATGGAGAGAAAATACAGAGGAAAGTGACCTGACCTCAGATAAAGCAGTGGGGTATATTACATCTGCAGTGCCCTACAGATCTGTATCATTGCTGCATTGTGACATTGAAATTCCTCCAGGGTCCAATCAAAGCTTACTAGATCAACTAGCTATCAATTTATCTCTGATTGCCATGCATTCTGTAAGGGAGGGCGGGGTAGTAATCATCAAAGTGTTGTATGCAATGGGATACTACTTTCATCTACTCATGAACTTGTTTGCTCCGTGTTCCACAAAAGGATATATTCTCTCTAATGGTTATGCATGTCGAGGAGATATGGAGTGTTACCTGGTATTTGTCATGGGTTACCTGGGCGGGCCTACATTTGTACATGAGGTGGTGAGGATGGCAAAAACTCTGGTGCAGCGGCACGGTACGCTCTTGTCTAAATCAGATGAGATCACACTGACCAGGTTATTCACCTCACAGCGGCAGCGTGTGACAGACATCCTATCCAGTCCTTTACCAAGATTAATAAAGTACTTGAGGAAGAATATTGACACTGCGCTGATTGAAGCCGGGGGACAGCCCGTCCGTCCATTCTGTGCGGAGAGTCTGGTGAGCACGCTAGCGAACATAACTCAGATAACCCAGATTATCGCTAGTCACATTGACACAGTTATCCGGTCTGTGATATATATGGAAGCTGAGGGTGATCTCGCTGACACAGTATTTCTATTTACCCCTTACAATCTCTCTACTGACGGGAAAAAGAGGACATCACTTATACAGTGCACGAGACAGATCCTAGAGGTTACAATACTAGGTCTTAGAGTCGAAAATCTCAATAAAATAGGCGATATAATCAGCCTAGTGCTTAAAGGCATGATCTCCATGGAGGACCTTATCCCACTAAGGACATACTTGAAGCATAGTACCTGCCCTAAATATTTGAAGGCTGTCCTAGGTATTACCAAACTCAAAGAAATGTTTACAGACACTTCTGTATTGTACTTGACTCGTGCTCAACAAAAATTCTACATGAAAACTATAGGCAATGCAGTCAAAGGATATTACAGTAACTGTGACTCTTAACGAAAATCACATATTAATAGGCTCCTTTTTTGGCCAATTGTATTCTTGTTGATTTAATCATATTATGTTAGAAAAAAGTTGAACCCTGACTCCTTAGGACTCGAATTCGAACTCAAATAAATGTCTTAAAAAAAGGTTGCGCACAATTATTCTTGAGTGTAGTCTCG(Part d) TCATTCACCAAATCTTTGTTTGGT

[0109] TABLE 4 BC_CAT_.TXT (Part a)ACCAAACAGAGAATCCGTAAGTTACGATAAAAGGCGAAGGAGCAATTGAAGTTGCACGGGTAGAAGGTGTGAATCTCGAGTGCGAGCCCGAAGCACAAACTCGAGAAAGCCTTCTGCCAACATGTCTTCCGTATTTGACGAGTACGAACAGCTCCTCGCGGCTCAGACTCGCCCCAATGGAGCTCATGGAGGAGGGGAAAAGGGGAGTACCTTAAAAGTAGACGTCCCGGTATTCACTCTTAACAGTGATGACCCAGAAGATAGGTGGAACTTTGCGGTATTCTGCCTCCGGATTGCTGTTAGCGAAGATGCCAACAAACCACTCAGGCAAGGTGCTCTCATATCTCTTTTATGCTCCCACTCACAAGTGATGAGGAACCATGTTGCCCTTGCAGGGAAACAGAATGAAGCCACATTGGCCGTGCTTGAGATTGATGGCTTTGCCAACGGTATGCCCCAGTTCAACAATAGGAGTGGAGTGTCTGAAGAGAGAGCACAGAGATTCGCGATGATAGCAGGGTCTCTCCCTCGGGCATGCAGTAATGGCACCCCGTTCGTCACAGCCGGGGCCGAAGATGATGCACCAGAAGATATCACCGATACCCTGGAGAGGATCCTCTCTATCCAGGCCCAAGTATGGGTCACAGTAGCAAAAGCCATGACTGCGTATGAGACTGCAGATGAGTCTGAAACAAGACGAATCAGTAAGTATATGCAGCAAGGCAGGGTCCAAAAGAAATACATCCTCTACCCCGTATGCAGGAGCACAATCCAACTCACGATCAGACAGTCTCTTGCAGTCCGCATCTTTTTGGTTAGCGAGCTCAAGAGAGGCCGCAACACGGCAGGTGGTACCTCTACTTATTATAACCTAGTAGGGGACGTAGACTCATATATCAGGAATACCGGGCTTACTGCATTCTTCTTGACACTCAAGTACGGAATTAACACCAAGACATCAGCCCTTGCACTTAGTAGCCTCTCAGGCGACATCCAGAAAATGAAGCAGCTCATGCGTTTATATCGGATGAAAGGAGATAATGCGCCGTACATGACATTGCTTGGTGATAGTGACCAGATGAGCTTTGCGCCTGCCGAGTATGCACAACTTTACTCCTTCGCCATGGGTATGGCATCAGTCCTAGATAAAGGTACTGGGAAATACCAATTTGCCAGGGACTTTATGAGCACATCATTCTGGAGACTTGGAGTAGAGTACGCTCAGGCTCAGGGAAGTAGCATTAACGAGGATATGGCTGCCGAGCTAAAGTTAACCCCAGCAGCAAGGAGAGGCCTGGCAGCTGCTGCCCAACGAGTCTCTGAGGAGACCAGCAGCATAGACATGCCTACTCAACAAGTCGGAGTCCTCACTGGGCTCAGCGAGGGGGGGTCCCAAGCCCTACAAGGCGGATCGAATAGATCGCAAGGGCAACCAGAAGCCGGGGATGGGGAGACCCAATTCCTGGATCTGATGAGAGCGGTAGCAAATAGCATGAGGGAAGCGCCAAACTCTGCACAGGGCACTCCCCAATCGGGGCCTCCCCCAACTCCTGGGCCATCTCAAGATAACGACACCGACTGGGGGTATTGATTGACAAAACCCAGCTTGCTTCCACAAAATCATCCCAATATCCTCACCCGTAGTCGACCCCTCGATTTGCGGCCCTATATGACCACACCCACAAACAAACATCCCCCTCTTTCCTCCCTCCCCCTGCTGTACAACTCCGCACGCCCTAGGTACCACAGGCACAATGCGGCTCACTAACAATCAAAACAGAGCCGAGGAAATTAGAAAAAAATACGGGTAGAAGAGGGATATTCAGAGACCAGGGCAAGTCACCCGAGTCTCTGCTCTCTCCTCTACCTGATAGATTAGGACAAATATGGCCACCTTTACAGATGCGGAGATCGACGAGCTATTTGAGACAAGTGGAACTGTGATTGACAACATAATTACAGCCCAGGGTAAATCAGCAGAGACTGTTGGAAGGAGTGCAATCCCACATGGCAAAACCAAGGCGCTGAGCGCAGCATGGGAGAAGCATGGGAGCATCCAGCCACCGGCCAGTCAAGACACCCCTGATCGACAGGACAGATCTGACAAACAACCATCCACACCCGAGCAAGCGACCCCGCATGACAGCCCGCCGGCCACATCCGCCGACCAGCCCCCCACCCAGGCCACAGACGAAGCCGTCGACACACAGCTCAGGACCGGAGCAAGCAACTCTCTGCTGTTGATGCTTGACAAGCTCAGCAATAAATCGTCCAATGCTAAAAAGGGCCTATGGTCGAGCCCCCAAGAGGGGAACCACCAACGTCCGACTCAACAGCAGGGAAGTCAACCCAGCCGCGGAAACAGCCAGGAAAGACCGCAGAACCAAGTCAAGGCCGCCCCTGGAAACCAGGGCACAGACGCGAACACAGCATATCATGGACAATGGGAGGAGTCACAACTATCAGCTGGTGCAACCCCTCATGCTCTCCGATCAAGGCAGAGCCAAGACAATACCCTTGTATCTGCGGATCATGTCCAGCCACCTGTAGACTTTGTGCAAGCGATGATGTCTATGATGGAGGCAATATCACAGAGAGTAAGTAAGGTTGACTATCAGCTAGATCTTGTCTTGAAACAGACATCCTCCATCCCTATGATGCGGTCCGAAATCCAACAGCTGAAAACATCTGTTGCAGTCATGGAAGCCAATTTGGGAATGATGAAGATTCTGGATCCCGGTTGTGCCAACGTTTCATCTCTGAGTGATCTACGGGCAGTTGCCCGATCTCACCCGGTTTTAGTTTCAGGCCCTGGAGACCCATCTCCCTATGTGACTCAAGGAGGCGAAATGGCACTTAATAAACTTTCGCAACCAGTGCCACATCCATCTGAATTGATTAAATCCGCCACTGCATGCGGGCCTGATATAGGAGTGGAAAAGGACACTGTCCGTGCATTGATCATGTCACGCCCAATGCACCCGAGTTCTTCAGCCAAGCTCCTAAGCAAGCTAGATGCAGCCGGGTCGATCGAGGAAATCAGGAAAATCAAGCGCCTTGCACTAAATGGCTAATTACTACTGCCACACGTAGCGGGTCCCCGTCCACTCGGCATCACACGGAATCTGCACCGAGTCCCCCCCCGCAGACCTAAGGTCCAACTCTCCAAGTGGCAATCCTCTCTCGCTTCCTCAGCCCCACTGAATGATCGCGCAACCGTAATTAATCTAGCTACATTAAGGATTAAGAAAAAATACGGGTAGAATTGGAGTGCCCCAATTGTGCCAAGATGGACTCATCTAGGACAATTGGGCTGTACTTTGATTCTGCCCATTCTTCTAGCAACCTGTTAGCATTTCCGATCGTCCTACAAGACACAGGAGATGGGAAGAAGCAAATCGCCCCGCAATATAGGATCCAGCGCCTAGACTCGTGGACTGATAGTAAAGAAGACTCAGTATTCATCACCACCTATGGATTCATCTTTCAGGTTGGGAATGAAGAAGCCACTGTCGGCATGATCAATGATAATCCCAAGCGCGAGTTACTTTCTGCTGCGATGCTCTGCCTAGGAAGCGTCCCAAATACCGGAGACCTTGTTGAGCTGGCAAGGGCCTGTCTCACTATGGTAGTCACATGCAAGAAGAGTGCAACTAATACTGAGAGAATGGTTTTCTCAGTAGTGCAGGCACCCCAAGTGCTGCAAAGCTGTAGGGTTGTGGCAAACAAATACTCATCAGTGAATGCAGTCAAGCACGTGAAAGCGCCAGAGAAGATCCCCGGGAGTGGAACCCTAGAAIACAAGGTGAACTTTGTCTCCTTGACTGTGGTACCGAAGAAGGATGTCTACAAGATCCCAACTGCAGTATTGAAGGTTTCTGGCTCGAGTCTGTACAATCTTGCGCTCAATGTCACTATTAATGTGGAGGTAGACTCGAGGAGTCCTTTGGTTAAATCTCTGTCTAAGTCTGACAGCGGAIACTATGCTAACCTCTTCTTGCATATTGGACTTATGACCACCGTAGATAGGAGGGGGAAGAAAGTGACTTTTGACAAGCTAGAAAAGAAGATAAGGAGCCTTGATCTATCTGTCGGGCTCAGTGATGTGCTCGGACCTTCCGTGCTGGTAAAAGCAAGAGGTGCACGGACCAAGCTTTTGGCACCTTTCTTCTCTAGCAGTGGGACAGCCTGCTATCCCATAGCAAATGCCTCTCCTCAGGTGGCCAAGATACTCTGGAGTCAAACCGCGTGCCTGCGGAGCGTTAAAATCATTATCCAAGCAGGTACCCAACGCACCGTCGCAGTGACCGCTGACCACGAGGTTACCTCTACTAAGCTGGAGAAGGGGCACACCCTTGCCAAATACAATCCTTTTAAGAAATAAGCTGCGTCTCTGAGATTGCGCTCCGCCCACTCACCCAGAGCATCATGACACCAAAAACTAATCTGTCTTGATTATTTACAGTTAGTTTACCTGTCTATCAAATTAGAAAAAACACGGGTAGAAGATTCTGGATCCCGGTTGGCGCCTTCTAGGTGCAAGATGGGCCCCAGACCTTCTACCAAGAACCCAGTACCTATGATGCTGACTGTCCGAGTCGCGCTGGTACTGAGTTGCATCTGTCCGGCAAACTCCATTGATGGCAGGCCTCTTGCGGCTGCAGGAATTGTGGTAACAGGAGACAAAGCAGTCAACATATACACCTCATCCCAGACAGGATCAATCATAGTTAAGCTCCTCCCAAACCTGCCCAAGGATAAGGAGGCATGTGCGAAAGCCCCCTTGGATGCATACAACAGGACATTGACCACTTTGCTCACCCCCCTTGGTGACTCTATCCGTAGGATACAAGAGTCTGTAACTACATCTGGAGGGAGGAGACAGAAACGCTTTATAGGCGCCATTATTGGCGGTGTGGCTCTTGGGGTTGCAACTGCTGCACAAATAACAGCGGCCGCAGCTCTGATACAAGCCAAACAAAATGCTGCCAACATCCTCCGACTTAAAGAGAGCATTGCCGCAACCAATGAGGCCGTGCATGAGGTCACTGACGGATTATCGCAACTAGCAGTGGCAGTTGGGAAGATGCAGCAGTTTGTTAATGACCAATTTAATAAAACAGCTCAGGAATTAGGCTGCATCAGAATTGCACAGCAAGTTGGTGTAGAGCTCAACCTGTACCTAACCGAATTGACTACAGTATTCGGACCACAAATCACTTCACCTGCCTTAAACAAGCTGACTATTCAGGCACTTTACAATCTAGCTGGTGGGAATATGGATTACTTGTT(Part b)GACTAAGTTAGGTGTAGGGAACAATCAACTCAGCTCATTAATCGGTAGCGGCTTAATCACCGGCAACCCTATTCTGTACGACTCACAGACTCAACTCTTGGGTATACAGGTAACTCTACCTTCAGTCGGGAACCTAAATAATATGCGTGCCACCTACTTGGAAACCTTATCCGTAAGCACAACCAGGGGATTTGCCTCGGCACTTGTCCCAAAAGTGGTGACACAGGTCGGTTCTGTGATAGAAGAACTTGACACCTCATATTGTATAGAAACCGACTTGGATTTATATTGTACAAGAATAGTAACATTCCCTATGTCCCCTGGTATTTATTCCTGCTTGAGCGGCAATACATCGGCCTGTATGTACTCAAAGACCGAAGGCGCACTCACTACGCCATACATGACTATCAAAGGCTCAGTCATCGCTAACTGCAAGATGACAACAIGTAGATGTGTAAACCCCCCGGGTATCATATCGCAAAACTATGGAGAAGCCGTGTCTCTAATAGATAAGCAATCATGCAATGTTTTATCCTTAGACGGGATAACTTTAAGGCTCAGTGGGGAATTCGATGCAACTTATCAGAAGAATATCTCAATACAAGATTCTCAAGTAATAATAACAGGCAATCTTGATATCTCAACTGAGCTTGGGAATGTCAACAACTCGATCAGTAATGCTTTGAATAAGTTAGAGGAAAGCAACAGCAAACTAGACAAAGTCAATGTCAAACTGACCAGCACATCTGCTCTCATTACCTATATCGTTTTGACTATCATATCTCTTGTTTTTGGTATACTTAGCCTGGTTCTAGCATGCTACCTAATGTATAAGCAAAAGGCGCAACAAAAGACCTTATTATGGCTTGGGAATAATACCCTAGATCAGATGAGAGCCACTACAAAAATGTGAACACAGATGAGGAACGAAGGTATCCCIAATAGTAATTTGTGIGAAAGTTCTGGTAGTCTGTCAATTCGGAGAGTTTAGAAAAAACTACGCGTTGTAGATGACCAAAGGACGATATACGGGTAGAACGGTAAGAGAGGCCGCCCCTCAATTGCGAGCCGGGCTTCACAACCTCCGTTCTACCGCTTCACCGACAGCAGTCCTCAGTCATGGACCGCGCAGTTAGCCAAGTTGCGTTAGAGAATGATGAAAGAGAGGCAAAAAATACATGGCGCTTGATATTCCGGATTGCAATCTTACTCTTAACAGTAGTGACCTTAGCTACATCTGTAGCCTCCCTTGTATATAGCATGGGGGCTAGCACACCTAGCGACCTTGTAGGCATACCGACCAGGATTTCTAGGGCAGAAGAAAAGATTACATCTGCACTTGGTTCCAATCAAGATGTAGTAGATAGGATATATAAGCAAGTGGCCCTTGAGTCTCCGTTGGCATTGTTAAACACTGAGACCACAATTATGAACGCAATAACATCTCTCTCTTATCAGATTAATGGAGCTGCGAACAACAGCGGGTGGGGGGCACCTATCCATGACCCAGATTTTATCGGGGGGATAGGCAAAGAACTCATTGTAGATGATGCTAGTGATGTCACATCATTCTATCCCTCTGCATTTCAAGAACATCTGAATTTTATCCCGGCGCCTACTACAGGATCAGGTTGCACTCGGATACCTTCATTTGACATGAGTGCTACCCATTACTGCTACACTCATAATGTAATATTGTCTGGATGCAGAGATCACTCACACTCACATCAGTATTTAGCACTTGGTGTGCTCCGGACAACTGCAACAGGGAGGATATTCTTTTCTACTCTGCGTTCCATCAGTCTGGATGACACCCAAAATCGGAAGTCTTGCAGTGTGAGTGCAACTCCCTTAGGTTGTGATATGCTGTGCTCGAAAGTCACGGAGACAGAGGAAGAAGATTATAACTCAGCTGTCCCTACGCTGATGGCACATGGGAGGTTAGGGTTCGACGGCCAATACCACGAAAAGGACCTAGACGTCACAACATTATTTGAGGACTGGGTGGCCAACTACCCAGGAGTAGGGGGTGGATCTTTTATTGACGGCCGCGTATGGTTCTCAGTCTACGGAGGGCTGAAACCCAATTCACCCAGTGACACTGTACAGGAAGGGAAATATGTAATATACAAGCGATACAATGACACATGCCCAGATGAGCAAGACTACCAGATCCGAATGGCCAAGTCTTCGTATAAGCCCGGGCGGTTTGGTGGGAAACGCATACAGCAGGCTATCTTATCTATCAAGGTGTCAACATCTTTGGGCGAAGACCCAGTACTGACTGTACCGCCCAACACAGTCACACTCATGGGGGCCGAAGGCAGAATTCTCACAGTAGGGACATCTCATTTCTTGTATCAGCGAGGGTCATCATACTTCTCTCCCGCGTTATTATATCCTATGACAGTCAGCAACAAAACAGCCACTCTTCATAGTCCCTATACATTCAATGCCTTCACTCGGCCAGGTAGTATCCCTTGCCAGGCTTCAGCAAGATGCCCCAACTCGTGTGTTACTGGAGTCTATACAGATCCATATCCCCTAATCTTCTATAGGAACCACACCTTGCGAGGGGTATTCGGGACAATGCTTGATAGTGAACAAGCAAGACTTAATCCTGCGTCTGCAGTATTCGATAGCACATCCCGCAGTCGCATAACTCGAGTGAGTTCAAGCAGCACCAAAGCAGCATACACAACATCAACTTGTTTTAAAGTTGTCAAGACCAATAAGACCTATTGTCTCAGCATTGCTGAAATATCTAATACTCTCTTCGGAGAATTCAGAATCGTCCCGTTACTAGTTGAGATCCTCAAAAATGATGGGG1TAGAGAAGCCAGGTCTGGTTAGTTGAGTCAACTATGAAAGAGCTGGGAAGATGGCATTGTATCACCTATCTTCCGCGACACCAAGAATCAAACTGAATGCCGGTGCGAGCTCGAATTCCATGTCGCCAGTTGACCACAATCAGCCAGTGCTCATGCGATCAGATCAAGTCTTGTCAATAGTCCCTCGATTAAGAAAAAATGTAAGTGGCAATGAGATACAAGGCAAAACAGCTACCGGTACGGGTAGAACGCCACCATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATACTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATGATTAGAAAAAAACCGGTAAATAGTACGGGTAGGACATGGCGAGCTCCGGTCCTGAAAGGGCAGAGCATCAGATTATCCTACCAGAGTCACACCTGTCTTCACCATTGGTCAAGCACAAACTACTTTATTACTGGAAATTAACTGGGCTACCGCTTCCTGATGAATGTGACTTCGACCACCTCATTCTCAGCAGACAATGGAAAAAAATACTTGAATCGGCCTCTCCTGATACTGAGAGAATGATAAAACTCGGAAGGGCAGTACACCAAACTCTCAACCACAATTCTAGAATAACCGGAGTACTCCACCCCAGGTGTTTAGAAGAACTGGCTAGTATTGAGGTCCCTGATTCAACCAACAAATTTCGGAAGATTGAGAAGAAGATCCAAATTCACAACACGAGATATGGAGAACTGTTCACAAGGCTGTGTACGCATATAGAGMGAAACTGCTGGGGTCATCCTGGTCTAACAATGTCCCCCGGTCAGAGGAGTTCAACAGCATCCGTACGGATCCGGCATTCTGGTTTCACTCAAAATGGTCCACAGCCAAGTTTGCATGGCTCCATATAAAACAGATCCAGAGGCATCTGATTGTGGCAGCTAGGACAAGGTCTGCGGCCAACAAATTGGTGATGCTAACCCATAAGGTAGGCCAAGTCTTTGTCACTCCTGAACTTGTCATTGTGACGCATACGAATGAGAACAAGTTCACATGTCTTACCCAGGAACTTGTATTGATGTATGCAGATATGATGGAGGGCAGAGATATGGTCAACATAATATCAACCACGGCGGTGCATCTCAGAAGCTTATCAGAGAAAATTGATGACATTTTGCAGTTAATAGACGCTCTGGCAAAAGACTTGGGCAATCAAGTCTACGATGTTGTATCACTAATGGAGGGATTTGCATACGGAGCTGTCCAGCTGCTCGAGCCGTCAGGTAGATTTGCAGGACATTTCTTCGCATTCAACCTGCAGGAGCTTAAAGACATTCTAATCGGCCTCCTCCCCAATGATATAGCAGAATCCGTGACTCATGCAATAGCTACTGTATTCTCTGGTTTAGAACAGAATCAAGCAGCTGAGATGTTGTGCCTGTTGCGTCTGTGGGGTCACCCACTGCTTGAGTCCCGTATTGCAGCAAAGGCAGTCAGGAGCCAAATGTGCGCACCGAAAATGGTGGAC1TTGATATGATCCTTCAGGTACTGTCTTTCTTCAAGGGAACAATCATCAACGGATACAGAAAGAAGAATGCAGGTGTGTGGCCGCGAGTCAAAGTGGATACAATATATGGGAAGATCATTGGGCAACTACATGCAGATTCAGCAGAGATTTCACACGATATCATGTTGAGAGAGTATAAGAGTTTATCTGCACTTGAATTTGAGCCATGTATAGAATACGACCCTGTCACTAACCTGAGCATGTTCCTAAAAGACAAGGCAATCGCACACCCTAACGATAATTGGCTTGCCTCGTTTAGGCGGAACCTTCTCTCCGAAGACCAGAAGAAACATGTAAAAGAAGCAACTTCGAC(Part c)TAATCGCCTCTTGATAGAGTTTTTAGAGTCAAATGATTTTGATCCATATAAAGAGATGGAATATCTGACGACCCTIGAGTACCTTAGAGATGACGATGTGGCAGTATCATACTCGCTCAAAGAGAAGGAAGTGAAAGTTAATGGACGGATCTTCGCTAAGCTGACAAAGAAGTTAAGGAACTGTCAGGTGATGGCGGAAGGGATCCTAGCCGACCAGATTGCACCTTTCTTTCAGGGAAATGGAGTCATTCAGGATAGCATATCGTTGACCAAGAGTATGCTAGCGATGAGTCAACTGTCTTTTAACAGCAATAAGAAACGTATCACTGACTGTAAAGAAAGAGTATCTTCAAACCGCAATCATGATCCGAAGAGCAAGAACCGTCGGAGAGTTGCAACCTTCATAACAACTGACCTGCAAAAGTACTGTCTTAATTGGAGATATCAGACAATCAAACTGTTCGCTCATGCCATCAATCAGTTGATGGGCCTACCTCACTTCTTTGAGTGGATTCACCTAAGACTGATGGACACTACAATGTTCGTAGGAGACCCTTTCAATCCTCCAAGTGACCCTACTGACTGTGACCTCTCAAGAGTCCCTAATGATGACATATATATTGTCAGTGCCAGAGGGGGTATCGAAGGATTATGTCAGAAGCTATGGACAATGATCTCTATTGCTGCAATCCAACTTGCTGCAGCTAGATCGCATTGTCGCGTTGCCTGTATGGTACAGGGTGATAATCAAGTAATAGCAGTAACGAGAGAGGTAAGATCAGACGACTCTCCGGAGATGGTGTTGACACAGTTGCATCAAGCCAGTGATAATTTCTTCAAGGAATTAATTCATGTCAATCATTTGATTGGCCATAATTTGAAGGACCGTGAAACCATCAGGTCAGACACATTCTTCATATACAGCAAACGAATCTTCAAAGATGGAGCAATCCTCAGTCAAGTCCTCAAAAATTCATCTAAATTAGTACTGGTGTCAGGTGATCTCAGTGAAAACACCGTAATGTCCTGTGCCAACATTGCCTCTACTGTAGCACGGCTATGCGAGAACGGGCTTCCCAAGGACTTCTGTTACTATTTAAACTATATAATGAGTTGCGTGCAGACATACTTTGACTCTGAGTTCTCCTACAACAACAATTCGCACCCCGATCTTAACCAGTCGTGGATTGAGGACATCTCTTTTGTGCACTCATATGTTCTGACTCCTGCCCAATTAGGGGGACTTAGTAACCTTCAATACTCAAGGCTCTACACTAGAAATATCGGTGACCCGGGGACTACTGCTTTTGCAGAGATCAAGCGACTAGAAGCAGTGGGATTACTGAGTCCTAACATTATGACTAATATCTTAACTAGGCCGCCTGGGAATGGAGATTGGGCCAGTCTTTGCAACGACCCATACTCTTTCAATTTTGAGACTGTTGCAAGCCCAAACATTGTTCTTAAGAAACATACGCAAAGAGTCCTATTTGAAACTTGTTCAAATCCCTTATTGTCTGGAGTGCACACAGAGGATAATGAGGCAGAAGAGAAGGCATTGGCTGAATTCTTGCTTAATCAAGAGGTGATTCATCCCCGCGTTGCGCATGCTATCATGGAGGCAAGCTCTGTAGGTAGGAGAAAGCAAATTCAAGGGCTTGTTGACACAACAAACACCGTAATTAAGATTGCACTTACTAGGAGGCCACTAGGCATCAAGAGGCTGATGCGGATAGTCAATTATTCTAGCATGCATGCAATGCTGTTTAGAGACGATGTTTTTTCCTCCAATAGAICCAACCACCCCTTAGTCTCTTCTAATATGTGTTCTCTGACACTGGCAGACTATGCACGGAATAGAAGCTGGTCACCTTTGACGGGAGGCAGGAAAATACTGGGTGTATCTAATCCTGATACGATAGAACTCGTAGAGGGTGAGATTCTTAGTGTAAGCGGAGGGTGCACAAGATGCGACAGCGGAGATGAACAGTTTACTTGGTTCCATCTTCCAAGCAATATAGAATTGACCGATGACACCAGCAAGAATCCTCCGATGAGAGTACCATATCTCGGGTCAAAGACACAGGAGAGGAGAGCTGCCTCACTTGCGAAAATAGCTCATATGTCGCCACATGTGAAGGCTGCCCTAAGGGCATCATCCGTGTTGATCTGGGCTTATGGGGATAATGAAGTAAATTGGACTGCTGCTCTTACGATTGCAAAATCTCGATGTAATATAAACTTAGAGTATCTTCGGTTATTGTCCCCTTTACCCACGGCTGGGAATCTTCAACATAGACTAGATGATGGTATAACTCAGATGACATTCACCCCTGCATCICTCTACAGGGTGTCACCTTACATTCACATATCCAATGATTCTCAAAGGCTATTCACTGAAGAAGGAGTCAAAGAGGGGAATGTGGTTTATCAACAGATCATGCTCTTGGGTTTATCTCTAATCGAATCGATCTTTCCAATGACGACAACCAGGACATATGATGAGATCACATTGCATCTACATAGTAAATTTAGTTGCTGTATCAGGGAAGCACCTGTTGCGGTTCCTTTCGAGCTACTTGGGGTGGCACCGGAGCTAAGGACAGTGACCTCAAACAAGTTTATGTATGATCCTAGCCCTGTATCGGAGGGAGACTTTGCGAGACTTGACTTAGCTATCTTCAAGAGTTATGAGCTTAATCTGGAGTCATATCCCACGATAGAGCTAATGAACATTCTTTCAATATCCAGCGGGAAGTTGATTGGCCAGTCTGTGGTTTCTTATGATGAAGATACCTCCATAAAGAATGACGCCATAATAGTGTATGACAATACCCGAAATTGGATCAGTGAAGCTCAGAATTCAGATGTGGTCCGCTTATTTGAATATGCAGCACTTGAAGTGCTCCTCGACTGTTCTTACCAACTCTATTATCTGAGAGTAAGAGGCCTAGACAATATTGTCTTATATATGGGTGATTTATACAAGAATATGCCAGGAATTCTACTTTCCAACATTGCAGCTACAATATCTCATCCCGTCATTCATTCAAGGTTACATGCAGTGGGCCTGGTCAACCATAACGGATCACACCAACTTGCAGATACGGATTTTATCGAAATGTCTGCAAAACTGTTAGTATCTTGCACTCGACGTGTGATCTCCGGCTTATATTCAGGGAATAAGTATGATCTGCTGTTCCCATCTGTCTTAGATGATAACCTGAATGAGAAGATGCTTCAGCTGATATCCCGGTTATGCTGTCTGTACACGGTACTCTTTGCTACAACAAGAGAAATCCCGAAAATAAGAGGCTTATCTGCAGAAGAGAAATGTTCAGTACTTACTGAGTATCTACTGTCGGATGCTGTGAAACCATTACTTAGCCCTGATCAGGTGAGCTCTATCATGTCTCCTAACATAATTACATTCCCAGCTAATCTGTACTACATGTCTCGGAAGAGCCTCAATTTGATCAGGGAAAGGGAGGACAAGGATTCTATCCTGGCGTTGTTGTTCCCCCAAGAGCCATTATTAGAGTTCCCTTCTGTGCAAGATATTGGTGCTCGAGTGAAAGATCCATTCACCCGACAACCTGCGGCATTTTTGCAAGAGTTAGATTTGAGTGCTCCAGCAAGGTATGACGCATTCACACTTAGTCAGATTCATCCTGAGCTCACATCACCAAATCCGGAGGAAGACTACTTAGTACGATACTTGTTCAGAGGAATAGGGACTGCATCCTCCTCTTGGTATAAGGCATCCCATCTCCTTTCTGTACCCGAGGTAAGATGTGCAAGACACGGGAACTCCTTATACTTAGCTGAAGGAAGCGGAGCCATCATGAGTCTTCTCGAACTGCATGTACCACATGAAACTATCTATTACAATACGCTCTTTTCAAATGAGATGAACCCCCCGCAGCGACATTTCGGGCCGACCCCAACCCAGTTTTTGAATTCGGTTGTTTATAGGAACCTACAGGCGGAGGTAACATGCAAGGATGGATTTGTCCAAGAGTTCCGTCCACTATGGAGAGAAAATACAGAGGAAAGCGACCTGACCTCAGATAAAGCAGTGGGGTATATTACATCTGCAGTGCCCTACAGATCTGTATCATTGCTGCATTGTGACATTGAAATCCCTCCAGGGTCCAATCAAAGCTTACTAGATCAATTAGCTATCAATTTATCTCTGATTGCCATGCATTCCGTAAGGGAGGGCGGGGTAGTGATCATCAAAGTGTTGTATGCAATGGGATACTACTTTCATCTACTCATGAACTTGTTCGCTCCGTGTTCCACAAAAGGATACATTCTCTCTAATGGTTATGCATGTAGAGGGGATATGGAGTGTTACCTGGTATTTGTCATGGGTTACCTGGGCGGGCCTACATTTGTACACGAGGTGGTGAGGATGGCAAAAACTCTGGTGCAGCGGCACGGTACGCTTTTGTCCAAATCAGATGAGATCACACTGACCAGGTTATTCACCTCACAGCGGCAGCGTGTGACAGACATCCTATCCAGTCCTTTACCAAGATTAATAAAGTACTTGAGAAAGAATATTGACACTGCGCTGATTGAAGCTGGGGGACAGCCCGTCCGTCCATTCTGTGCAGAGAGTTTGGTGAGCACGCTGGCGGACATAACTCAGATAACCCAGATCATTGCTAGTCACATTGACACAGTCATCCGGTCTGTGATATATATGGAAGCTGAGGGTGATCTCGCTGACACAGTATTTCTATTTACCCCTTACAATCTCTCTACTGACGGGAAAAAGAGAACATCACTTAAACAGTGCACGAGACAGATCCTAGAGGTTACAATATTGGGTCTTAGAGTCGAAGATCTCAATAAAATAGGCGATGTAATCAGCCTAGTGCTTAAAGGCATGATCTCCATGGAGGACCTTATCCCACTAAGGACATACTTGAAGCATAGTACCTGCCCTAAATATTTGAAGGCTGTCCTAGGTATTACCAAACTCAAAGAAATGTTTACAGACACCTCTGTATTGTACTTGACTCGTGCTCAACAAAAATTCTACATGAAAACTATAGGCAATGCAGTCAAAGGATATTACAGTAACTGTGACTCTTAACGAAAATCACATATTAATAGGCTCCTTTTCTGGCCAATTGTATCCTTGGTGATTTAATTATACTATGTTAGAAAAAAATTGAACTCCGACTCCTTAGATCTCGAATTCGAACTCAAATAAATGTCTTAAAAAAAGGTTGCGCACAATTATTCTTGAGTGTAGTCTTGTTATTCACCAAATCTTTG(Part d) TTTGGT

1. An antigenomic RNA of Newcastle disease virus, comprising NP gene, Pgene, M gene, F gene, HN gene and L gene in this order from a 5′ to 3′direction, said antigenomic RNA further comprising n foreign nucleotidecomplexes inserted (a) before the NP gene, (b) between the P and Mgenes, and/or (c) between the HN and L genes, wherein n is 1, 2, 3 or 4;each of the foreign nucleotide complexes comprising a Newcastle diseasevirus gene start sequence, an open reading frame of a foreign gene and aNewcastle disease virus gene end sequence in this order from the 5′ to3′ direction, wherein the foreign gene is a gene not found naturally inthe Newcastle disease virus; and wherein when 2, 3 or 4 foreignnucleotide complexes are inserted together before the NP gene, betweenthe P and M genes, or between the HN and L genes, the foreign nucleotidecomplexes are sequentially linked directly or indirectly.
 2. Theantigenome RNA of claim 1, wherein n is 2, 3 or 4 and the foreignnucleotide complexes are different.
 3. The antigenome RNA of claim 1,wherein n is 2, 3 or 4 and the foreign nucleotide complexes are thesame.
 4. The antigenome RNA of claim 1, wherein n is 1 or
 2. 5. Theantigenome RNA of claim 4, wherein n is 2 and the foreign nucleotidecomplexes are different.
 6. The antigenome RNA of claim 4, wherein n is2 and the foreign nucleotide complexes are the same.
 7. The antigenomeRNA of claim 1, wherein the length of the open reading frame of theforeign gene is no more than about 3000 nucleotides.
 8. The antigenomeRNA of claim 7, wherein the length of the open reading frame of theforeign gene is no more than about 1500 nucleotides.
 9. The antigenomeRNA of claim 8, wherein the length of the open reading frame of theforeign gene is no more than about 1000 nucleotides.
 10. The antigenomeRNA of claim 9, wherein the length of the open reading frame of theforeign gene is no more than about 800 nucleotides.
 11. The antigenomeRNA of claim 10, wherein the length of the open reading frame of theforeign gene is no more than about 500 nucleotides.
 12. The antigenomeRNA of claim 11, wherein the length of the open reading frame of theforeign gene is no more than about 300 nucleotides.
 13. The antigenomeRNA of claim 1, wherein 2, 3 or 4 foreign nucleotide complexes areinserted together before the NP gene, between the P and M genes, orbetween the HN and L genes, the sequentially linked foreign nucleotidecomplexes having a combined length of no more than about 5000nucleotides.
 14. The antigenomic RNA of claim 13, wherein thesequentially linked foreign nucleotide complexes have a combined lengthof no more than about 2000 nucleotides.
 15. The antigenomic RNA of claim14, wherein the sequentially linked foreign nucleotide complexes have acombined length of no more than about 1000 nucleotides.
 16. Theantigenomic RNA of claim 15, wherein the sequentially linked foreignnucleotide complexes have a combined length of no more than about 800nucleotides.
 17. The antigenomic RNA of claim 1, wherein the foreigngenes of the foreign nucleotide complexes are selected from the groupconsisting of genes encoding chloramphenical acetyltransferase, greenfluorescent protein, an avian cytokine, and an immunogenic protein of avirus selected from the group consisting of influenza virus, infectiousbursal disease virus, rotavirus, infectious bronchitis virus, infectiouslaryngotracheitis virus, chicken anemia virus, Marek's disease virus,avian leukosis virus, avian adenovirus, and avian pneumovirus.
 18. Theantigenomic RNA of claim 1, wherein the foreign genes of the foreignnucleotide complexes encode chloramphenical acetyltransferase.
 19. Theantigenomic RNA of claim 1, wherein the foreign genes of the foreignnucleotide complexes encode the same or different avian cytokines. 20.The antigenomic RNA of claim 19, wherein the avian cytokines are avianinterleukins.
 21. The antigenomic RNA of claim 20, wherein the aviancytokines are avian IL-2 and/or IL-4.
 22. The antigenomic RNA of claim1, wherein the foreign genes of the foreign nucleotide complexes encodean immunogenic protein of the same or different viruses selected fromthe group consisting of influenza virus, infectious bursal diseasevirus, rotavirus, infectious bronchitis virus, infectiouslaryngotracheitis virus, chicken anemia virus, Marek's disease virus,avian leukosis virus, avian adenovirus and avian pneumovirus.
 23. Theantigenomic RNA of claim 1, wherein the n foreign nucleotide complexesare inserted (a) before the NP gene, and/or (b) between the P and Mgenes.
 24. The antigenomic RNA of claim 23, wherein the n foreignnucleotide complexes are inserted before the NP gene.
 25. The antigenomeRNA of claim 24, wherein 2, 3 or 4 foreign nucleotide complexes areinserted together before the NP gene, the sequentially linked foreignnucleotide complexes having a combined length of no more than about 4000nucleotides.
 26. The antigenomic RNA of claim 25, wherein thesequentially linked foreign nucleotide complexes have a combined lengthof no more than about 2000 nucleotides.
 27. The antigenomic RNA of claim26, wherein the sequentially linked foreign nucleotide complexes have acombined length of no more than about 1000 nucleotides.
 28. Theantigenomic RNA of claim 27, wherein the sequentially linked foreignnucleotide complexes have a combined length of no more than about 800nucleotides.
 29. The antigenomic RNA of claim 23, wherein the n foreignnucleotide complexes are inserted between the P and M genes.
 30. Theantigenomic RNA of claim 29, wherein the sequentially linked foreignnucleotide complexes have a combined length of no more than about 4000nucleotides.
 31. The antigenomic RNA of claim 30, wherein thesequentially linked foreign nucleotide complexes have a combined lengthof no more than about 2000 nucleotides.
 32. The antigenomic RNA of claim31, wherein the sequentially linked foreign nucleotide complexes have acombined length of no more than about 1000 nucleotides.
 33. Theantigenomic RNA of claim 32, wherein the sequentially linked foreignnucleotide complexes have a combined length of no more than about 800nucleotides.
 34. The antigenomic RNA of claim 18, wherein n is
 1. 35.The antigenomic RNA of claim 19, wherein n is
 1. 36. The antigenomic RNAof claim 22, wherein n is
 1. 37. The antigenomic RNA of claim 24,wherein n is
 1. 38. The antigenomic RNA of claim 37, wherein the openreading frame of the foreign gene encodes chloramphenicolacetyltransferase.
 39. The antigenomic RNA of claim 37, wherein the openreading frame of the foreign gene encodes an avian cytokine.
 40. Theantigenomic RNA of claim 39, wherein the avian cytokine is an avianinterleukin.
 41. The antigenomic RNA of claim 40, wherein the avianinterleukin is IL-2 or IL-4.
 42. The antigenomic RNA of claim 1, havinga length of m nucleotides, wherein m is a multiple of
 6. 43. Theantigenomic RNA of claim 1, wherein at least one of the foreignnucleotide complexes contain foreign gene encoding an immunogenicprotein of a non-avian pathogen.
 44. The antigenomic RNA of claim 43,wherein the non-avian pathogen is a virus selected from the groupconsisting of influenza virus, SARS-causing virus, human respiratorysyncytial virus, human immunodeficiency virus, hepatitis A virus,hepatitis B virus, hepatitis C virus, poliovirus, rabies virus, Hendravirus, Nipah virus, human parainfluenza 3 virus, measles virus, mumpsvirus, Ebola virus, Marburg virus, West Nile virus, Japaneseencephalitis virus, Dengue virus, Hantavirus, Rift Valley fever virus,Lassa fever virus, herpes simplex virus and yellow fever virus.
 45. AcDNA encoding the antigenomic RNA of claim
 1. 46. A plasmid comprisingthe cDNA of claim
 45. 47. A cell comprising the cDNA of claim
 45. 48. Acell comprising the plasmid of claim
 46. 49. A cell comprising theantigenomic RNA of claim
 1. 50. A recombinant Newcastle disease virusproduced by a process comprising the following steps: (i) providingcells capable of synthesizing T7 RNA polymerase; (ii) transfecting thecells with a plasmid comprising the cDNA encoding the antigenomic RNA ofclaim 1, a plasmid encoding NP protein, a plasmid encoding P protein,and a plasmid encoding L protein to obtain transfected cells in amedium; and thereafter (iii) isolating Newcastle disease virus from asupernatant of the medium of step (ii) to obtain the recombinantNewcastle disease virus.
 51. The recombinant Newcastle disease virus ofclaim 50, wherein the cells capable of synthesizing T7 RNA polymeraseprovided in step (i) are from a cell line expressing T7 RNA polymerase.52. The recombinant Newcastle disease virus of claim 50, wherein thecells capable of synthesizing T7 RNA polymerase provided in step (i) areplant cells.
 53. The recombinant Newcastle disease virus of claim 50,wherein the cells capable of synthesizing T7 RNA polymerase provided instep (i) are mammalian cells.
 54. The recombinant Newcastle diseasevirus of claim 50, wherein the cells capable of synthesizing T7 RNApolymerase provided in step (i) are avian cells.
 55. The recombinantNewcastle disease virus of claim 50, wherein the cells capable ofsynthesizing T7 RNA polymerase provided in step (i) are HEp-2 cellsinfected with a virus that can synthesize T7 RNA polymerase.
 56. Therecombinant Newcastle disease virus of claim 55, wherein the virus is avaccinia virus.
 57. A recombinant Newcastle disease virus produced by aprocess comprising the following steps: (i) providing cells capable ofsynthesizing T7 RNA polymerase; (ii) transfecting the cells with aplasmid comprising the cDNA encoding the antigenomic RNA of claim 18, aplasmid encoding NP protein, a plasmid encoding P protein, and a plasmidencoding L protein to obtain transfected cells in a medium; andthereafter (iii) isolating Newcastle disease virus from a supernatant ofthe medium of step (ii) to obtain the recombinant Newcastle diseasevirus.
 58. A recombinant Newcastle disease virus produced by a processcomprising the following steps: (i) providing cells capable ofsynthesizing T7 RNA polymerase; (ii) transfecting the cells with aplasmid comprising the cDNA encoding the antigenomic RNA of claim 19, aplasmid encoding NP protein, a plasmid encoding P protein, and a plasmidencoding L protein to obtain transfected cells in a medium; andthereafter (iii) isolating Newcastle disease virus from a supernatant ofthe medium of step (ii) to obtain the recombinant Newcastle diseasevirus.
 59. A recombinant Newcastle disease virus produced by a processcomprising the following steps: (i) providing cells capable ofsynthesizing T7 RNA polymerase; (ii) transfecting the cells with aplasmid comprising the cDNA encoding the antigenomic RNA of claim 22, aplasmid encoding NP protein, a plasmid encoding P protein, and a plasmidencoding L protein to obtain transfected cells in a medium; andthereafter (iii) isolating Newcastle disease virus from a supernatant ofthe medium of step (ii) to obtain the recombinant Newcastle diseasevirus.
 60. A recombinant Newcastle disease virus produced by a processcomprising the following steps: (i) providing cells capable ofsynthesizing T7 RNA polymerase; (ii) transfecting the cells with aplasmid comprising the cDNA encoding the antigenomic RNA of claim 43, aplasmid encoding NP protein, a plasmid encoding P protein, and a plasmidencoding L protein to obtain transfected cells in a medium; andthereafter (iii) isolating Newcastle disease virus from a supernatant ofthe medium of step (ii) to obtain the recombinant Newcastle diseasevirus.
 61. A method of vaccinating an avian animal against Newcastledisease, wherein the avian animal is in need of the vaccination,comprising administering an effective amount of the recombinantNewcastle disease virus of claim 50 to the avian animal.
 62. A method ofvaccinating an avian animal against Newcastle disease, wherein the aviananimal is in need of the vaccination, comprising administering aneffective amount of the recombinant Newcastle disease virus of claim 57to the avian animal.
 63. A method of treating an avian animal with anavian cytokine, wherein the avian animal is in need of the treatment,said method comprising administering an effective amount of therecombinant Newcastle disease virus of claim 58 to the avian animal. 64.A method of immunizing an avian animal against an avian pathogenselected from the group consisting of influenza virus, infectious bursaldisease virus, rotavirus, infectious bronchitis virus, infectiouslaryngotracheitis virus, chicken anemia virus, Marek's disease virus,avian Leukosis virus, avian adenovirus and avian pneumovirus, whereinthe avian animal is in need of the immunization, said method comprisingadministering an effective amount of the recombinant Newcastle diseasevirus of claim 59 to the avian animal, wherein the open reading frame ofthe foreign gene encodes an immunogenic protein of the avian pathogenagainst which the avian animal is immunized.
 65. A method of immunizinga mammal against a non-avian pathogen, wherein the mammal is in need ofthe immunization, said method comprising administering an effectiveamount of the recombinant Newcastle disease virus of claim 60 to themammal, wherein the open reading frame of the foreign gene encodes animmunogenic protein of the non-avian pathogen against which the mammalis immunized.
 66. The method of claim 65, wherein the non-avian pathogenis selected from the group consisting of influenza virus, SARS-causingvirus, human respiratory syncytial virus, human immunodeficiency virus,hepatitis A virus, hepatitis B virus, hepatitis C virus, poliovirus,rabies virus, Hendra virus, Nipah virus, human parainfluenza 3 virus,measles virus, mumps virus, Ebola virus, Marburg virus, West Nile virus,Japanese encephalitis virus, Dengue virus, Hantavirus, Rift Valley fevervirus, Lassa fever virus, herpes simplex virus and yellow fever virus.67. A process of making the antigenomic RNA of claim 1, comprising thefollowing steps: (i) providing a cDNA comprising NP gene, P gene, Mgene, F gene, HN gene and L gene in this order, said cDNA furthercomprising n foreign nucleotide complexes inserted (a) before the NPgene, (b) between the P and M genes, and/or (c) between the HN and Lgenes, wherein n is 1, 2, 3 or 4; each of the foreign nucleotidecomplexes comprising a Newcastle disease virus gene start sequence, anopen reading frame of a foreign gene and a Newcastle disease virus geneend sequence in this order from the 5′ to 3′ direction, wherein theforeign gene is a gene not found naturally in the Newcastle diseasevirus; wherein when n is 2, 3 or 4, the foreign nucleotide complexes arethe same or different; and wherein when 2, 3 or 4 foreign nucleotidecomplexes are inserted together before the NP gene, between the P and Mgenes, or between the HN and L genes, the foreign nucleotide complexesare sequentially linked directly or indirectly; (ii) transcribing thecDNA to form a mixture containing an antigenomic RNA; and thereafter(iii) isolating the antigenomic RNA.
 68. The antigenomic RNA of claim 1,wherein at least one of the foreign nucleotide complexes is insertedbefore the NP gene.
 69. The antigenomic RNA of claim 1, wherein at leastone of the foreign nucleotide complexes is inserted before the NP geneand at least one of the foreign nucleotide complexes is inserted betweenthe P and M genes.
 70. The antigenomic RNA of claim 1, wherein at leastone of the foreign nucleotide complexes is inserted before the NP geneand at least one of the foreign nucleotide complexes is inserted betweenthe HN and L genes.
 71. The antigenomic RNA of claim 1, wherein at leastone of the foreign nucleotide complexes is inserted before the NP gene,at least one of the foreign nucleotide complexes is inserted between theP and M genes, and at least one of the foreign nucleotide complexes isinserted between the HN and L genes.
 72. The antigenomic RNA of claim 1,wherein at least one of the foreign nucleotide complexes is insertedbetween the P and M genes.
 73. The antigenomic RNA of claim 1, furthercomprising at least one intergenic region selected from the groupconsisting of a NP-P intergenic region between the NP and P genes, a P-Mintergenic region between the P and M genes, a M-F intergenic regionbetween the M and F genes, a F-HN intergenic region between the F and HNgenes, and a HN-L intergenic region between the HN and L genes.
 74. Theantigenomic RNA of claim 1, further comprising a NP-P intergenic regionbetween the NP and P genes, a P-M intergenic region between the P and Mgenes, a M-F intergenic region between the M and F genes, a F-HNintergenic region between the F and HN genes, and a HN-L intergenicregion between the HN and L genes.
 75. The antigenomic RNA of claim 1,wherein the foreign gene of at least one of the foreign nucleotidecomplexes encodes a tumor antigen.
 76. The antigenomic RNA of claim 75,wherein the tumor antigen is selected from the group consisting ofpg100, MAGE1, MAGE3 and CDK4.
 77. A recombinant Newcastle disease viruscomprising the antigenomic RNA of claim
 1. 78. A recombinant Newcastledisease virus comprising the antigenomic RNA of claim
 18. 79. Arecombinant Newcastle disease virus comprising the antigenomic RNA ofclaim
 19. 80. A recombinant Newcastle disease virus comprising theantigenomic RNA of claim
 22. 81. A recombinant Newcastle disease viruscomprising the antigenomic RNA of claim
 43. 82. A method of vaccinatingan avian animal against Newcastle disease, wherein the avian animal isin need of the vaccination, comprising administering an effective amountof the recombinant Newcastle disease virus of claim 77 to the aviananimal.
 83. A method of vaccinating an avian animal against Newcastledisease, wherein the avian animal is in need of the vaccination,comprising administering an effective amount of the recombinantNewcastle disease virus of claim 78 to the avian animal.
 84. A method oftreating an avian animal with an avian cytokine, wherein the aviananimal is in need of the treatment, said method comprising administeringan effective amount of the recombinant Newcastle disease virus of claim79 to the avian animal.
 85. A method of immunizing an avian animalagainst an avian pathogen selected from the group consisting ofinfluenza virus, infectious bursal disease virus, rotavirus, infectiousbronchitis virus, infectious laryngotracheitis virus, chicken anemiavirus, Marek's disease virus, avian Leukosis virus, avian adenovirus andavian pneumovirus, wherein the avian animal is in need of theimmunization, said method comprising administering an effective amountof the recombinant Newcastle disease virus of claim 80 to the aviananimal, wherein the open reading frame of the foreign gene encodes animmunogenic protein of the avian pathogen against which the avian animalis immunized.
 86. A method of immunizing a mammal against a non-avianpathogen, wherein the mammal is in need of the immunization, said methodcomprising administering an effective amount of the recombinantNewcastle disease virus of claim 81 to the mammal, wherein the openreading frame of the foreign gene encodes an immunogenic protein of thenon-avian pathogen against which the mammal is immunized.
 87. Theantigenomic RNA of claim 1, wherein at least one foreign nucleotidecomplex is inserted between the P and M genes and at least one foreignnucleotide complex is inserted before the NP gene.
 88. The antigenomicRNA of claim 1, wherein at least one foreign nucleotide complex isinserted between the P and M genes and at least one foreign nucleotidecomplex is inserted between the HN and L genes.